InteractiveFly: GeneBrief

Tsc1: Biological Overview | References

BIOLOGICAL OVERVIEW

FOXO is thought to function as a repressor of growth that is, in turn, inhibited by insulin signaling. However, inactivating mutations in Drosophila melanogaster FOXO result in viable flies of normal size, which raises a question over the involvement of FOXO in growth regulation. Previously, a growth-suppressive role for FOXO under conditions of increased target of rapamycin (TOR) pathway activity was described. This study further characterizes this phenomenon. Tuberous sclerosis complex 1 mutations cause increased FOXO levels, resulting in elevated expression of FOXO-regulated genes, some of which are known to antagonize growth-promoting pathways. Analogous transcriptional changes are observed in mammalian cells, which implies that FOXO attenuates TOR-driven growth in diverse species (Harvey, 2008).

Recent studies in mammals have revealed the molecular mechanism for the tumor suppressor function of TSC1 and TSC2, which form a physical and functional complex. The TSC1-TSC2 complex suppresses cell growth by inhibiting the mammalian target of rapamycin, mTOR, which is a central controller of cell growth. TSC1/TSC2 has GTPase-activating protein (GAP) activity toward the Rheb small GTPase. Rheb acts upstream of and stimulates mTOR. TSC2 is the catalytic GAP subunit, while TSC1 enhances TSC2 function by stabilizing TSC2. The majority of disease-associated TSC1 mutations identified result in no TSC1 protein being expressed; therefore, the free TSC2 protein in TSC1 mutant cells is unstable. Similarly, many disease-derived TSC2 mutants are unstable due to weakened interaction with TSC1. A potential mechanism be which TSC1 stabilizes TSC2 is the exclusion of the HERC1 ubiquitin ligase from the TSC2 complex (Chong-Kopera, 2006 and references therein).

To investigate mechanisms by which the TOR pathway controls tissue growth, transcriptional profiles were analyzed of tissue lacking Tsc1, which leads to hyperactivation of the TOR pathway and excessive growth. Eye-antennal imaginal discs from third instar Drosophila larvae were generated that were composed almost entirely of tissue derived from one of two different genotypes: Tsc1 or wild-type isogenic control. Three biologically independent first strand cDNA samples from each genotype were hybridized to microarray chips. Expression levels of 157 genes were elevated 1.5-fold or more, whereas 211 genes were repressed 1.5-fold or more when compared with control tissue. These genes have been implicated in diverse cellular functions including metabolism, membrane transport, stress response, cell growth, and cell structure (Harvey, 2008).

Observed transcriptional changes were validated for several genes using Drosophila gene-enhancer trap lines. The UAS-Gal4 system was used to activate the TOR pathway in a specific tissue domain by driving expression of Rheb under the control of the glass multiple reporter (GMR) promoter. Induction of astray (aay) and 4E-BP (both of which were found to be elevated in Tsc1 tissue by microarray analysis) were observed in the GMR expression domain (posterior to the morphogenetic furrow) when Rheb was misexpressed but were not induced when the negative control Gal4 gene was misexpressed. QPCR was also used to confirm expression changes observed in Tsc1 tissue for charybdis (chrb), scylla (scy), phosphoenolpyruvate carboxy kinase, 4E-BP, and aay (Harvey, 2008).

Intriguingly, several gene products whose expression was elevated in Tsc1 tissue have been implicated in tissue growth controlled by the insulin and TOR pathways, including 4E-BP, Chrb, and Scy. 4E-BP is a repressor of cap-dependent translation. Upon phosphorylation by TOR, 4E-BP dissociates from eIF4E, allowing assembly of the initiation complex at the mRNA cap structure, ribosome recruitment, and subsequent translation. Scy and Chrb, and their mammalian orthologues REDD1 and REDD2, inhibit insulin and TOR signaling in response to hypoxia and energy stress and restrict growth during Drosophila development. The finding that inhibitors of growth are highly expressed in Tsc1 tissue led to the hypothesis that such genes are transcriptionally induced as part of a feedback loop that restricts tissue growth under conditions of excessive TOR activity. Feedback loops are an important activity-modulating feature of many signaling pathways, including the TOR and insulin pathways (Harvey, 2008).

To examine the mechanism whereby transcription of growth inhibitors is induced in response to TOR hyperactivation, attempts were made to determine which transcription factors were responsible for their expression. One obvious candidate was FOXO, a member of the forkhead transcription factor family, which has a well-established role as an effector of insulin signaling. If FOXO has a role in inducing expression of negative regulators of growth in Tsc tissue, then expression of some of those genes should be elevated under conditions of increased FOXO activity. To investigate this hypothesis, the expression profiles were examined of Tsc1 LOF tissue and Drosophila S2 cells expressing FOXOA3, a mutant version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt. This analysis revealed that 25 genes were up-regulated 1.5-fold or greater in both Tsc1 LOF and FOXO GOF expression profiles, which represents a highly significant degree of overlap as determined by calculation of the hypergeometric distribution. A highly statistically significant P value strongly suggests that there is a functional overlap between these two datasets that cannot be explained by random variation (Harvey, 2008).

Interestingly, two genes previously implicated in tissue growth regulated by the insulin and TOR pathways 4E-BP and scy were elevated in both microarray experiments, whereas the chrb growth-inhibiting gene was not. Thus, a subset of genes elevated in Tsc1 tissue appears to respond to FOXO activity and was investigated further (Harvey, 2008).

4E-BP is a well-characterized FOXO target gene. To determine whether FOXO could directly activate transcription of genes that were elevated in Tsc1 tissue other than 4E-BP, focus was placed on scy and the phosphoserine phosphatase aay (one of the most highly elevated transcripts in each microarray experiment). scy and aay both possess consensus FOXO recognition elements (FREs) in their promoters comparable to those found in dInR and 4E-BP promoters. Therefore, whether these genes are bona fide FOXO targets was examined by measuring their expression in Drosophila S2 cells misexpressing FOXOA3 in the presence of insulin. aay and scy mRNAs were up-regulated 19.4- and 4.3-fold, respectively, relative to a control gene, actin, as determined by QPCR (Harvey, 2008).

Luciferase reporter assays in S2 cells were used to determine whether the aay promoter region containing putative FREs was sensitive to FOXO activity. Luciferase activity dependent on the aay promoter was strongly induced by FOXOA3. In addition, using in vitro band shift assays, it was demonstrated that FOXO directly binds to the aay promoter, indicating that FOXO likely activates expression of aay by directly binding to the FRE. Surprisingly, in parallel luciferase reporter assays, activation of the scy promoter by FOXO could not be demonstrated, despite the fact that strong binding of FOXO to the putative scy FRE was observed using in vitro band-shift assays. A possible explanation is that the scy-promoter construct lacked the minimal promoter elements required for transcription of luciferase (Harvey, 2008).

TOR pathway hyperactivation caused by Tsc deficiency has been shown to strongly repress activity of Akt. FOXO is normally inactivated by Akt-dependent phosphorylation, which restricts nuclear entry of FOXO and leads to its ubiquitin-dependent destruction. Therefore, in response to TOR pathway hyperactivation, it was predicted that reduced Akt activity would cause FOXO protein to accumulate. To examine this hypothesis, expression of FOXO protein was analyzed in mosaic Tsc1 imaginal discs. It was found that FOXO protein was markedly increased in Tsc1 clones when compared with neighboring wild-type tissue (Harvey, 2008).

In addition, FOXO protein appeared to be mostly nuclear in Tsc1 tissue and cytoplasmic in wild-type tissue. Consistent with this observation, nuclear localization of the mouse FOXO orthologue FOXO1 is observed in endothelial cells of Tsc2 mutant hemangiomas, whereas FOXO1 is mostly cytoplasmic in normal cells. FOXO mRNA levels are unchanged in Tsc1 tissue as determined by microarray analysis, which suggests that changes in translation or stability of FOXO protein account for its accumulation in Tsc1 tissue. The presence of increased FOXO protein in the nuclei of Tsc1 cells is consistent with the hypothesis that FOXO is responsible for increased expression of some of the growth inhibitors that are up-regulated in Tsc1 cells (Harvey, 2008).

To determine whether FOXO was necessary for transcriptional induction of genes that were elevated in Tsc1 tissue, QPCR analysis was used to measure 4E-BP, aay, and scy expression in Tsc1 and Tsc1-FOXO double mutant eye-antennal imaginal discs. Consistent with microarray analysis, increased expression of 4E-BP, aay, and scy was observed in Tsc1 tissue. In Tsc1-FOXO tissue, however, 4E-BP was expressed at approximately equivalent amounts as in wild-type tissue, whereas aay and scy expression was only partially reduced. This demonstrates that elevated expression of 4E-BP in Tsc1 tissue is dependent on the FOXO transcription factor and provides evidence that FOXO activity increases when the TOR pathway is hyperactivated. Expression of aay and scy appear to be partially dependent on FOXO but are likely stimulated by additional transcription factors in Tsc1 tissue (Harvey, 2008).

Next, attempts were made to determine whether FOXO is required to limit growth of tissues with increased TOR pathway activity. In addition, a potential role was examined for another transcription factor, HIF-1, for retardation of TOR-driven growth. HIF-1 is a dual-subunit transcription factor consisting of α and β subunits that functions in response to insulin/TOR signaling and drives transcription of the growth-inhibiting genes scy and chrb, both of which are elevated in Tsc1 tissue (Harvey, 2008).

Drosophila possesses several HIF-1α subunits and a sole HIF-1β subunit, tango (tgo), which partners with each HIF-1α subunit. If FOXO and/or HIF-1 are required to induce expression of genes that limit tissue growth when the TOR pathway is hyperactivated, one might predict that Tsc1-FOXO and/or Tsc1-tgo double mutant tissue would possess a greater capacity to grow than Tsc1 tissue alone. To test this hypothesis, the size was examined of Drosophila eyes comprised almost entirely of the following genotypes: control, tgo, FOXO, Tsc1, Tsc1-tgo, and Tsc1-FOXO. Mutant eyes were created by driving mitotic recombination of chromosomes bearing flipase recognition target (FRT) sites and the appropriate gene mutations, specifically in developing Drosophila eye-antennal imaginal discs. Eyes lacking either tgo or FOXO were approximately the same size as control eyes, whereas Tsc1 eyes were considerably larger. Tsc1-tgo double mutant eyes did not exhibit a further increase in size, which suggests that HIF-1 is not required to inhibit tissue growth in response to Tsc1 loss. In contrast, Tsc1-FOXO double mutant eyes were substantially larger than Tsc1 eyes. This finding is particularly significant in light of the finding that eyes lacking FOXO were indistinguishable in size from wild-type eyes. Thus, it appears that FOXO is normally dispensable for control of eye size, but when growth control is altered by virtue of increased TOR activity, FOXO partially offsets the increased tissue growth. These findings are consistent with observations that FOXO protein accumulates in Tsc1 tissue and that transcriptional profiles of FOXO GOF and Tsc1 LOF cells overlap significantly (Harvey, 2008).

Because individual components of the insulin and TOR pathways are highly conserved among eukaryotes, important regulatory mechanisms that control tissue growth via these pathways are also likely to be conserved. To investigate this idea, transcriptional control was analyzed of mouse orthologues of genes that were elevated in D. melanogaster Tsc1 tissue. Initially, Northern blotting analysis was performed on Tsc2 primary mouse embryonic fibroblasts (MEFs; derived on a p53 background to overcome premature senescence induced by Tsc2 loss). It is reasonable to predict that transcriptional changes that occur because of loss of either Tsc1 or Tsc2 should be very similar because TSC1 and TSC2 function together in an obligate fashion, and mutation of either gene leads to almost indistinguishable phenotypes. It was found that several gene expression changes observed in Drosophila Tsc1 tissue are conserved in Tsc2 MEFs (Harvey, 2008).

The homologues of aay, heat shock protein (hsp) 23, scy, and chrb (PSPH, hsp 27, REDD1, and REDD2, respectively) were all significantly up-regulated in Tsc2 MEFs when compared with control MEFs and expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. To demonstrate that these expression changes were a specific consequence of Tsc2 loss, Tsc2 expression was reconstituted in Tsc2 null cells, which substantially suppressed mammalian TOR activity and expression of these genes. Interestingly, expression of phosphoenolpyruvate carboxy kinase and 4E-BP1/2 was not altered between wild-type and Tsc2 cells, which might reflect tissue- or species-specific differences in the transcriptome of Drosophila epithelial cells and MEFs (Harvey, 2008).

To determine whether the mode of transcription of these genes was also conserved in mammals, expression was analyzed of the scy homologue REDD1. Like scy, mammalian REDD1 orthologues possess a putative consensus FRE within their proximal promoters. Cotransfection of a version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt (TM-FKHRL-1) induced robust activation of a mouse REDD1 reporter construct in primary MEFs. To determine whether induction was mediated through the identified FRE, a mutant reporter was created lacking this sequence. Deletion of the REDD1 FRE consistently reduced FOXO-mediated induction of the REDD1 promoter. Finally, to directly assess whether FOXO-dependent transcription was activated in mammalian cells lacking Tsc2, activity of the REDD1 promoter reporter or the corresponding mutant FRE reporter was examined in wild-type and Tsc2 MEFs. As predicted, the wild-type REDD1 promoter exhibited robust activation in Tsc2 cells compared with wild-type cells, and this activation was substantially reduced by deletion of the FRE. Together, these findings provide evidence that transcriptional changes resulting from Tsc1/Tsc2 deficiency are conserved in diverse species (Harvey, 2008).

This study has identified of an evolutionary conserved transcriptional program important for restricting tissue overgrowth driven by excessive activation of the TOR pathway. The FOXO transcription factor plays a key role in this transcriptional response, likely by stimulating expression of several growth inhibitory genes. Thus, although the requirement for FOXO in restricting growth under normal development conditions appears dispensable, this is no longer the case under conditions of excessive TOR activation. These findings have important implications for cancer syndromes that arise because of inappropriate TOR pathway activation, such as the human hamartomatous syndrome, tuberous sclerosis. TOR-dependent feedback inhibition is thought to contribute to the benign nature of Tsc1 and Tsc2 tumors (Ma, 2005; Manning, 2005). Conceivably, inactivating mutations in FOXO family transcription factors and/or FOXO target genes that possess growth-inhibiting properties could promote further growth in normally benign Tsc1 and Tsc2 tumors (Harvey, 2008).

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).

Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells

Intestinal stem cells (ISCs) in the adult Drosophila melanogaster midgut can respond to damage and support repair. This study shows that the tuberous sclerosis complex (TSC) plays a critical role in balancing ISC growth and division. Previous studies have shown that imaginal disc cells that are mutant for TSC have increased rates of growth and division. However, this study shows that loss of TSC in the adult Drosophila midgut results in the formation of much larger ISCs that have halted cell division. These mutant ISCs express proper stem cell markers, do not differentiate, and have defects in multiple steps of the cell cycle. Slowing the growth by feeding rapamycin or reducing Myc is sufficient to rescue the division defect. The TSC mutant guts have a thinner epithelial structure than wild-type tissues, and the mutant flies are more susceptible to tissue damage. Therefore, this study has uncovered a context-dependent phenotype of TSC mutants in adult ISCs, such that the excessive growth leads to inhibition of division (Amcheslavsky, 2011).

This study provides evidence demonstrating that TSC is an essential regulator of ISC growth and division. In the absence of TSC function, ISCs have unrestricted cell growth, which halts cell division and leads to the formation of extremely large cells. Although stem cell markers are still expressed, these ISC-like cells are nonfunctional and can no longer divide or differentiate. As a consequence, the TSC mutant gut has a thinner epithelium and the mutant fly is more susceptible to tissue-damaging agents. This study has uncovered a tissue context-dependent phenotype of TSC mutants, such that unrestricted cell growth can lead to a stop of cell division, and thus, TSC does not function all the time as a classical tumor suppressor (Amcheslavsky, 2011).

The TSC-TOR and other growth regulatory pathways, such as InR and Myc, have intricate interactions. It has been suggested that the InR pathway directly represses TSC, whereas others and the current study suggest that the two pathways act in parallel. The TSC-TOR pathway also has a negative feedback into upstream components of the InR pathway. Recent identification of TORC2 in addition to the original TORC1 further complicates these pathways. However, the current results clearly show that TORC2 mutants and TSC mutants have different phenotypes in the adult Drosophila midgut, suggesting that TSC does not function through TORC2 to regulate ISC division. Previous studies have demonstrated that Myc can modulate TSC-TOR in controlling the growth of mammalian and fly cells, which is consistent with what this study has observed (Amcheslavsky, 2011).

In normal development and adult tissue homeostasis, cells need to grow in size by approximately twofold before they divide to maintain the original cell size. Reduction in cell growth below a certain threshold can lead to a halt of division. Therefore, the balance between cell growth versus division is a complex process requiring delicate coordination. This study has shown that, in TSC mutants, the increase in midgut ISC size is >10-fold, whereas the increase in larval disc cell size is less than twofold. A possible reason for this difference is that the mutant larval disc cells continue to divide, thereby maintaining a moderate cell size. One key question that remains is why the larval disc cells that contain a TSC mutation have somewhat coordinated growth and division, whereas the adult mutant ISCs have completely stopped their division. It is possible that because imaginal discs are developing organs, they are designed to have faster growth and division. Adult midgut ISCs have a slower intrinsic cell cycle of >24 h, and adult cells have differences in checkpoint controls. These may allow the excessive growth to take place until it passes a critical point that blocks division (Amcheslavsky, 2011).

Phenotypes manifested in TSC patients are mostly benign tumors that rarely progress into higher-grade cancers. TSC1 and 2 have expression in the intestine, and adult patients have occasional intestinal polyps. Mouse embryonic fibroblasts from mutant TSC animals can also enter senescence, which is equivalent to a cessation of cell division. The adult midgut ISC phenotypes shown in this study are consistent with these phenotypes. It is speculated that excessive cell growth leading to a block in cell division is a common phenotype in slowly dividing adult tissues when TSC is mutated. The phenotype of increased cell growth and increased cell division may be applicable to rapidly dividing cells, including developing Drosophila disc cells, mammalian hematopoietic stem cells, and tumor cells. A recent study demonstrates that in TSC mutants, there is loss of adult female germline stem cells because of differentiation. The ISCs and germline stem cells have different niche compositions that may contribute to the observed differences in the mutant phenotype. Moreover, it underscores the idea of a tissue context-dependent phenotype exhibited in TSC mutants (Amcheslavsky, 2011).

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).

Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase

Cellular growth and proliferation are coordinated during organogenesis. Misregulation of these processes leads to pathological conditions such as cancer. Tuberous sclerosis (TSC) is a benign tumour syndrome caused by mutations in either TSC1 or TSC2 tumour suppressor genes. Studies in Drosophila and other organisms have identified TSC signalling as a conserved pathway for growth control. Activation of the TSC pathway is mediated by Rheb (Ras homologue enriched in brain), a Ras superfamily GTPase. Rheb is a direct target of TSC2 Gigas in Drosophila) and is negatively regulated by its GTPase-activating protein activity. However, molecules required for positive regulation of Rheb have not been identified. This study shows that a conserved protein, translationally controlled tumour protein (TCTP), is an essential new component of the TSC-Rheb pathway. Reducing Drosophila TCTP (dTCTP) levels reduces cell size, cell number and organ size, which mimics Drosophila Rheb (dRheb) mutant phenotypes. dTCTP is genetically epistatic to Tsc1 and dRheb, but acts upstream of dS6k, a downstream target of dRheb. dTCTP directly associates with dRheb and displays guanine nucleotide exchange activity with it in vivo and in vitro. Human TCTP (hTCTP) shows similar biochemical properties compared to dTCTP and can rescue dTCTP mutant phenotypes, suggesting that the function of TCTP in the TSC pathway is evolutionarily conserved. These studies identify TCTP as a direct regulator of Rheb and a potential therapeutic target for TSC disease (Hsu, 2007).

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).

Mechanisms of TSC-mediated control of synapse assembly and axon guidance

Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, this study examined the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development (Knox, 2007).

The Tsc-Rheb-Tor pathway is critical for integrating a variety of signals that govern cellular and organismal growth. Inappropriate activation of the pathway also leads to severe neurological and behavioral abnormalities, including mental retardation, autism, and epilepsy. While TSC mutations produce hamartomatous growths in the brain, recent evidence has suggested that these benign tumors may not be solely responsible for the nervous system dysfunction that is a hallmark of tuberous sclerosis complex. Loss of TSC2 in hippocampal neurons produces changes in neuronal morphology and synaptic transmission. Heterozygosity for TSC2 in the rat compromises several measures of hippocampal long term potentiation. Loss of Pten, an important upstream regulator of Tsc-Rheb-Tor signaling, in a limited set of neurons also affects neuronal morphology and socialization behavior. These findings collectively provide evidence that Tsc-Rheb-Tor signaling is critical for the morphological and functional development of the nervous system. It is not clear, however, if the entire Tsc-Rheb-Tor signaling network is critical for nervous system development, or if neural function is strictly a consequence of altered growth regulation. It is also not known if loss of signaling is as detrimental to neuronal development as inappropriately elevated signaling, such as occurs with loss of TSC function. This study has taken advantage of the genetic and molecular tools available in Drosophila to address these questions. The findings demonstrate that appropriate levels of Tsc-Rheb-Tor signaling are critical for both NMJ development and for axon guidance in the visual system. In both these contexts, effects are independent of growth, implicating TORC2 (which includes Rictor in addition to Tor and mLST8; in both yeast and mammalian cells TORC2 influences the actin cytoskeleton) rather than TORC1 (which includes Raptor and mLST8, and regulates translation via phosphorylation of S6 kinase and 4E-binding protein) as the complex mediating Tsc-Rheb-Tor signaling influences in the nervous system (Knox, 2007).

Given the importance of Tsc-Rheb-Tor signaling in regulating cellular and tissue growth, it was important to determine if disruption of this pathway affects neural development via its effects on growth or through signaling components independent of those that govern cellular size and growth. To address this issue both pharmacological and genetic methods were used to block the increased growth produced by pathway activation. The immunosuppressant rapamycin is a TORC1-specific inhibitor that prevents activation of S6k and blocks growth mediated by loss of Tsc1. Rapamycin treatment retarded growth in larvae with pan-neuronal expression of Rheb, but failed to reduce the synapse expansion characteristic of these animals. Similarly, while rapamycin effectively reduced the retinal overgrowth of Tsc1 mosaic animals, it failed to suppress the photoreceptor axon guidance defects seen in the visual system. Loss of S6k function also failed to ameliorate axon guidance defects in Tsc1 mosaic animals. This contrasts with effects of Tor partial loss-of-function mutations, which effectively rescued axon guidance defects of Tsc1 mutants. Collectively, these findings demonstrate that the role of Tsc-Rheb-Tor signaling in synapse assembly and axon guidance is largely independent of TORC1, S6k, and their effects on growth. Indeed, while animals bearing null alleles of S6k have some axon pathfinding defects, the effects are relatively modest compared to Tsc1 mosaics, indicating that S6k does not provide the critical outputs affecting axon guidance (Knox, 2007).

The findings parallel recent work in the mouse, where neuronal hypertrophy produced by loss of Pten in granule neurons of the cerebellum and dentate gyrus was not rescued by loss of S6k1 (Chalhoub, 2006). It is also of note that some but not all Tsc1/2-mediated changes in dendritic morphology of hippocampal neurons in organotypic cultures were suppressed by rapamycin treatment (Tavazoie, 2005). The current findings suggest that inhibition of growth regulatory components in tuberous sclerosis patients, such as achieved with rapamycin and related agents, may not affect all processes that are deranged in the nervous system (Knox, 2007).

Recent studies of Pi3 kinase, Akt and InR in Drosophila have shown that activation of signaling upstream of Tsc1/2 also produces increases in synapse size, both at the NMJ as well as central synapses (Martin-Pena, 2006). Expression of these components in adult neurons demonstrated that Pi3 kinase-mediated synaptogenesis is age-independent, and therefore not a developmentally restricted phenomenon. In agreement with studies reported in this paper, the expanded NMJs produced by activation of Pi3 kinase were functional, with increased stimulus-induced EJPs. Overexpression of the Drosophila ortholog of the epidermal growth factor receptor (EgfR) in central neurons increased neuronal cell size, without an increase in synapse number. These results are consistent with those reported in this study it was possible to to directly suppress growth mediated by Tsc-Rheb-Tor pathway activation without altering effects on synapse formation or axon guidance (Knox, 2007).

Recent studies have also demonstrated a link between Tsc1/Tsc2 and highwire, a gene known to effect synapse size and functionality in Drosophila (Murthy, 2004). The highwire ortholog Pam was shown to bind Tsc2 in pull-down assays, and it has been suggested that Pam may function as an E3 ubiquitin ligase to regulate the intracellular levels of the Tsc1/Tsc2 complex. This concept of Highwire as a negative regulator of Tsc levels is consistent with the current findings, since highwire mutants have been shown to possess enlarged NMJs similar to what is seen for Rheb overexpression. Despite this, the enlarged synapses of highwire mutants display compromised synaptic function which is contrary to what was found when overexpressing Rheb, so Highwire is likely to have multiple functions at the synapse besides simply the regulation of Tsc (Knox, 2007).

Tor has a number of molecular outputs that influence many cellular processes; notable among these are cellular growth and cellular morphology. TORC1, which contains Raptor and is sensitive to the anti-proliferative agent rapamycin, is a major contributor to the regulation of cellular growth, in large measure due to its effects on protein synthesis. TORC2, which includes Rictor, is implicated in the control of cell morphology mediated by regulation of the actin cytoskeleton (Wullschleger, 2006). Both pharmacological and genetic studies presented here argue in favor of Tor complex 2 providing an essential regulatory component of both synapse growth and axon guidance in Drosophila. The current results support recent work showing that changes in dendritic morphology of hippocampal neurons produced by loss of Tsc1 required regulation of the actin-depolymerizing factor Cofilin (Tavazoie, 2005), implicating TORC2-mediated processes. There is a considerable body of work demonstrating that control of the actin cytoskeleton is critical for NMJ growth and function and TORC2 may provide an important component of that control. Regulation of actin is also essential for axon guidance in the visual system, and disruption of Tor-mediated control of actin may be the underlying molecular deficit in Tsc1 mosaics (Knox, 2007).

A number of studies have suggested that TOR activation produced by loss of TSC1/2 affects neuronal morphology and synaptic function. The current findings support these observations; elevated Rheb activity produces synaptic enlargement and enhanced physiological function at the Drosophila NMJ. However, it was not evident from earlier studies whether loss of signaling through Rheb and Tor is also important for neural development. Evidence is provided that this is the case. Partial loss-of-function mutations in Rheb compromise NMJ growth and function, as well as photoreceptor axon targeting in the visual system. Overexpression of Tsc1 and Tsc2 in the motoneuron also limited synaptic growth, supporting the conclusion that depressed levels of Rheb activity compromise synapse development (Knox, 2007).

The capacity of Tsc-Rheb-Tor signaling to affect neuronal morphology and synapse function begs the question of whether these effects are dependent on signaling systems known to be critical for synapse development. At the Drosophila NMJ, BMP signaling is critical for normal growth and function. Mutations in wit, a gene encoding a type II BMP receptor, produce a small and poorly functioning NMJ. These deficits can be rescued by motoneuron expression of wit⁺, demonstrating that BMP signaling in the motoneuron is critical for synaptic expansion during larval growth. To determine if Rheb-mediated synaptic growth requires BMP signaling, elav-Gal4 and UAS-Rheb transgenes were placed into a wit mutant background. While overexpression of Rheb and the accompanying activation of the Tor pathway partially rescued the defect in synapse growth produced by loss of wit function, it was unable to restore a normal EJP response or rescue quantal content. These findings establish that Tsc-Rheb-Tor mediated effects on synapse morphology are partially dependent on BMP signaling, and are fully dependent on BMP activity for a physiologically competent synapse. The findings also establish that the functional deficits in wit mutants are not simply the result of reduced synapse size, since restoration of synapse size by expression of UAS-Rheb does not restore physiological function. Intersection of BMP, and Akt/PTEN/TOR signaling has been noted for other systems, and the results indicate the relationship between these pathways is important for synapse growth and plasticity as well (Knox, 2007).

Previous analysis of gigas/Tsc2 mutants demonstrated that loss of this gene in mechanoreceptors affects axon targeting, producing projections to novel areas in the CNS in addition to innervation of normal targets (Canal, 1998). Genetic mosaics were used to evaluate the function of Tsc-Rheb-Tor signaling in photoreceptor axon guidance. Animals homozygous for Tsc1 in the retina showed grossly aberrant photoreceptor projections to both the lamina and medulla. R7 and R8 projections to the medulla in 40h pupae failed to terminate correctly and projected beyond normal targets to inappropriate regions within the brain. Somatic mosaics bearing retinal neurons mutant for Pten also showed photoreceptor axon guidance defects, but to a notably lesser degree. Since both Tsc1 and Pten alleles used for this analysis were nulls and show comparable effects on cellular growth and differentiation, it follows that Pten is not as critical for axon guidance as Tsc1. The distinctions between axon guidance phenotypes of Pten and Tsc1 null mutants indicate that altered timing of differentiation is not critical for axon guidance and that control of this pathway at the level of Pten or Tsc1 is not functionally equivalent. The findings that rapamycin arrests retinal overgrowth produced by loss of Tsc1 but not Pten in the retina supports earlier work demonstrating that retinal overgrowth mediated by loss of Tsc1, but not Pten, can be suppressed by reductions in S6k activity. Those results were interpreted as demonstrating that Pten is largely a regulator of Akt activity, whereas Tsc1/2 serves as a tumor suppressor and inhibitor affecting principally S6k. The current results support these relationships and emphasize that in the nervous system regulation of Tsc1/2 targets other than S6k are critical (Knox, 2007).

Two different genetic methods were used for activating the Tsc-Rheb-Tor pathway in the visual system; generating retinal mosaics with a loss of function allele of Tsc1, and pan-neuronal expression of Rheb using elav-Gal4 and UAS-Rheb. The comparison of these methods revealed that overexpression of Rheb produced milder axon guidance phenotypes in the visual system than complete loss of Tsc1 function. Of interest is that the degree of activation achieved with elav-Gal4>UAS-Rheb, a level that did not produce lethality, did result in discernable axon targeting defects in the visual system. This suggests that axon guidance controlled by Tsc-Rheb-Tor is sensitive to incremental changes in signaling. The range of neurological and behavioral phenotypes associated with loss of one copy of TSC1 or TSC2 is consistent with this model, where other environmental or genetic factors may affect signaling levels, producing a range of deficits. The findings indicate that Drosophila can serve as a useful model for identifying how graded changes in signaling can produce a spectrum of defects in neural development (Knox, 2007).

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. 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. 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).

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).

Rheb is a direct target of the tuberous sclerosis tumor suppressor proteins

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).

Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling

Target of Rapamycin (TOR) mediates a signalling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth. This study shows that Tuberous sclerosis 1 (Tsc1) and Tsc2, tumour suppressors that are responsible for the tuberous sclerosis syndrome, antagonize this amino acid-TOR signalling pathway. Tsc1 and Tsc2 can physically associate with TOR and function upstream of TOR genetically. In Drosophila and mammalian cells, loss of Tsc1 and Tsc2 results in a TOR-dependent increase of S6K activity. Furthermore, although S6K is normally inactivated in animal cells in response to amino acid starvation, loss of Tsc1-Tsc2 renders cells resistant to amino acid starvation. It is proposed that the Tsc1-Tsc2 complex antagonizes the TOR-mediated response to amino acid availability. These studies identify Tsc1 and Tsc2 as regulators of the amino acid-TOR pathway and provide a new paradigm for how proteins involved in nutrient sensing function as tumour suppressors (Gao, 2002).

In higher eukaryotes, cell growth is regulated by nutrient availability and growth factors. There is increasing evidence that nutrients, in particular amino acids, are sensed by a signalling pathway involving TOR (also known as mTOR/FRAP/RAFT1 in mammals), a Ser/Thr protein kinase that is specifically inhibited by the immunosuppressant rapamycin. This TOR-dependent amino acid signalling pathway has been shown to control downstream translational initiation regulators, including S6K and initiation factor 4E binding protein (4E-BP; also known as PHAS). Amino acid starvation results in a rapid dephosphorylation of S6K and 4E-BP, whereas re-addition of amino acids restores S6K and 4E-BP phosphorylation in a TOR-dependent manner. A major unanswered question in TOR signalling is how amino acids are sensed by cells and how such signals are transduced through TOR to downstream effectors. Through a parallel signalling pathway, growth factors, such as insulin or insulin-like growth factors, also regulate the activities of S6K and 4E-BP. The contribution of insulin and TOR signalling to cell growth has been demonstrated by recent genetic studies in Drosophila: down-regulation of either pathway results in decreased cell size, whereas up-regulation results in increased cell size (Gao, 2002).

Several negative regulators of cell growth have been identified in Drosophila, including Tsc1 and Tsc2. TSC1 and TSC2 were initially identified as tumour suppressor genes in humans. Mutations in TSC1 or TSC2 cause tuberous sclerosis, a syndrome that is characterized by the widespread development of benign tumours, termed harmatomas. TSC2 encodes a putative GTPase-activating protein (GAP), whereas TSC1 encodes a protein that contains two coiled-coil domains. TSC1 and TSC2 exist as a TSC1-TSC2 protein complex, such that mutations in one gene result in phenotypes that are identical to those caused by mutations in the other. The molecular function of the TSC1-TSC2 complex, however, has been elusive. Recent studies of Drosophila Tsc1 and Tsc2 homologues identified a specific function for Tsc1-Tsc2 in the control of cell growth, with a loss of Tsc1-Tsc2 resulting in an increase in cell size. These studies suggest that Tsc1-Tsc2 functions in a pathway parallel to insulin pathway, and that Tsc1-Tsc2 converges on the insulin pathway upstream of S6K. That the TOR-dependent amino acid signalling pathway converges on the insulin pathway at a similar position prompted examination a possible relationship between Tsc1-Tsc2 and TOR. First, it was tested whether there are any genetic interactions between Tsc1-Tsc2 and TOR. Flies homozygous for a null allele of Tsc1, Tsc1²⁹, die soon after hatching into first-instar larva. Strikingly, the lethality of Tsc1-null animals was partially rescued by removing one copy of the TOR gene from the diploid genome. Approximately 31% of Tsc1²⁹ homozygotes that were also heterozygous for TOR^2L1, a null allele of TOR, can survive to pupal stage. This represents an extension of viability by over five days. Consistent with such a dramatic rescue in whole-animal viability, heterozygosity for TOR significantly suppressed the increases in cell size that was observed in the Tsc1 mutant cells of mosaic eyes. Although homozygous Tsc1²⁹ mutant cells were 1.9 times the size of their wild-type neighbours, homozygous Tsc1²⁹ mutant cells that were also heterozygous for TOR^2L1 were only 1.3 times the size of their Tsc1⁺ neighbours. Thus, a 50% reduction in the dosage of the TOR gene, which normally does not affect cell size or the viability of the organism in a wild-type background, significantly rescued the developmental defect of Tsc mutants at the level of both the cell and the organism. Such dosage-sensitive interactions suggest that Tsc1-Tsc2 may be a previously unidentified component of the TOR signalling pathway. To further examine the epistatic relationship between Tsc1-Tsc2 and TOR, Tsc1-TOR double-mutant cells were generated and their cell-size phenotype were compared to that of cells lacking either Tsc1 or TOR. Although Tsc1 and TOR mutant cells are 1.9 and 0.26 times the size of wild-type cells, respectively, Tsc1-TOR double-mutant cells are 0.25 times the size of wild-type cells, comparable to the size of TOR single-mutant cells. This genetic epistasis result suggests that TOR functions downstream of, or in parallel to, Tsc1-Tsc2, in a signalling pathway that controls cell growth (Gao, 2002).

To explore further the dosage-sensitive genetic interactions between Tsc1-Tsc2 and TOR, whether Tsc1 or Tsc2 could physically associate with TOR was tested. Co-immunoprecipitation assays were performed in Drosophila Schneider 2 (S2) cells overexpressing epitope-tagged Tsc1 and TOR, or Tsc2 and TOR. These experiments identified a specific interaction between Tsc2 and TOR, and a specific, albeit weaker, interaction between Tsc1 and TOR. Furthermore, endogenous Tsc2 was specifically immunoprecipitated with epitope-tagged TOR. It was not possible to test the interactions between endogenous TOR and Tsc1-Tsc2 because of a lack of appropriate antibodies against Drosophila TOR. Furthermore, attempts to co-immunoprecipitate mammalian TOR (mTOR) with Tsc1-Tsc2 were inconclusive because of the poor specificity of the commercial antibodies tested. Although co-immunoprecipitation experiments involve overexpressed proteins, and thus are not necessarily indicative of an association of endogenous proteins in vivo, these results are nevertheless consistent with a model in which Tsc1-Tsc2 and TOR function in a common signalling pathway (Gao, 2002).

Next, it was asked if Tsc1-Tsc2 regulate the signalling activity of TOR. Although TOR possesses kinase activity that can be measured in TOR immunoprecipitates, this activity does not reflect its physiological regulation in most cases. The only available assay that accurately reflects the physiological regulation of TOR relies on measuring the activity of its downstream effectors, such as S6K. Thus, to examine the potential regulation of TOR by Tsc1-Tsc2, RNA interference (RNAi) was carried out to specifically knockout Tsc1 or Tsc2 in Drosophila S2 cells and analysed S6K activity using a phospho-specific antibody that detects activated S6K. RNAi of Tsc1 or Tsc2 resulted in a greater than 90% reduction of the respective proteins in S2 cells. These RNAi-treated cells are referred to as Tsc1(R) or Tsc2(R) cells. RNAi of Tsc2 resulted in a 4.5-fold increase in S6K phosphorylation, without a change in total S6K protein levels. RNAi of Tsc1 produced similar results. These observations provide direct evidence that Tsc1-Tsc2 negatively regulates the activity of S6K and are consistent with a recent report showing increased levels of phosphorylated S6K in Tsc1-null mouse fibroblasts. To determine if the regulation of S6K by Tsc1-Tsc2 is TOR-dependent, two experiments were performed. First, TOR activity was eliminated by treating cells with rapamycin, a specific inhibitor of TOR. Rapamycin abolished S6K activity in both Tsc2(R) and control cells. Second, TOR activity was eliminated with RNAi, which abolished S6K activity in both Tsc2(R) and control cells. These cell culture studies, together with the genetic epistasis analyses and the physical interactions between Tsc1-Tsc2 and TOR, suggest that the Tsc1-Tsc2 complex negatively regulates S6K activity, probably through TOR. Consistent with this hypothesis, it was found that the increase in size of Tsc2 mutant cells in mosaic eyes was partially suppressed by a null mutation of S6K. This partial suppression probably reflects the existence of additional effectors that are involved in cell growth, such as 4E-BP, which function downstream of TOR (Gao, 2002).

As mTOR is required in a signalling pathway that couples amino-acid levels to S6K activity, it was of interest to determine what relationship, if any, exists between Tsc1-Tsc2 and amino-acid signalling. It was found that in Drosophila S2 cells, an amino-acid signalling pathway operates to regulate S6K activity, as found in mammalian cells and Drosophila Kc167 cells. Switching wild-type S2 cells from complete medium to amino-acid-free medium caused a rapid dephosphorylation of S6K. As a result, S6K activity was reduced by 90% after 15 min and abolished after 30 min. Interestingly, it was found that Tsc2(R) cells were resistant to amino-acid starvation. In these cells, S6K activity was reduced by 39% after 30 min and remained at a similar level for an extended time. Even after 6 h of starvation, 35% of the S6K activity remained. A similar level of resistance to amino-acid starvation was also observed in Tsc1 and Tsc1-Tsc2 double-RNAi cells. That removal of Tsc1-Tsc2 blocks the effect of amino-acid starvation demonstrates that Tsc1-Tsc2 is normally involved in amino-acid signalling. The incomplete resistance of Tsc1-Tsc2(R) cells to amino-acid starvation could be a result of the incomplete knockout of Tsc1-Tsc2 by RNAi. It is worth noting that the resistance of Tsc1-Tsc2(R) cells to amino-acid starvation is a very specific phenotype: RNAi of PTEN (phosphatases with tensin domain), a negative regulator of the insulin pathway, resulted in an increase in S6K activity, as detected in Tsc1-Tsc2(R) cells. However, the PTEN(R) cells were as sensitive to amino-acid starvation as wild-type cells. In contrast to the different response of wild-type and Tsc2(R) cells to amino-acid starvation, both types of cells responded similarly to varying doses of rapamycin. Thus, loss of Tsc2 renders cells resistant to amino acid starvation without changing their sensitivity to rapamycin (Gao, 2002).

The resistance of Tsc1/2(R) cells to amino-acid starvation is probably caused by constitutive activation of the amino-acid-TOR pathway that is no longer dependent on amino acids. However, in an unlikely, but possible, scenario, loss of Tsc1-Tsc2 could result in an abnormally high concentration of intracellular amino acids that is sufficient to activate TOR, irrespective of extracellular amino-acid levels. To rule out the latter possibility, the concentration of intracellular amino acids was directly measured after amino-acid starvation and no differences were found between control and Tsc2(R) cells. Thus, Tsc1-Tsc2 is specifically involved in regulating signalling from amino acids to TOR, rather than in the synthesis, storage or metabolism of amino acids. Taken together, a model is favored in which Tsc1-Tsc2 negatively regulates the amino acid-TOR pathway and impinges on this pathway downstream of amino acids, but upstream of TOR. This model could explain the differential sensitivity of Tsc1/2(R) cells to amino-acid starvation and rapamycin: loss of Tsc1-Tsc2 causes constitutive activation of the amino acid-TOR pathway that no longer requires amino acids, but which still remains fully inhibited by rapamycin (Gao, 2002).

To investigate whether the requirement for Tsc1-Tsc2 in amino-acid signalling is applicable to all animals, the analyses of Tsc1-Tsc2 function were extended to mammalian cells. The Eker rat carries a germ-line insertion in the rat Tsc2 gene, resulting in truncation of the carboxyl terminus of the Tsc2 protein, and has been used extensively as an animal model for tuberous sclerosis. Previously, embryonic fibroblast cell lines have been derived from Tsc2^-/- and Tsc2^+/+ littermates. Using these cell lines (EEF4 for Tsc2^+/+ and EEF8 for Tsc2^-/-), the regulation of S6K by Tsc2 and its sensitivity to rapamycin and amino-acid starvation was examined. As observed in Drosophila cells, loss of Tsc2 results in a 5.4-fold increase in S6K activity in rat embryonic fibroblasts. In further agreement with the results from Drosophila cells, although Tsc2^+/+ rat cells were sensitive to both rapamycin and amino-acid starvation, Tsc2^-/- rat cells were sensitive to rapamycin but resistant to amino-acid starvation. It is worth noting Tsc2-negative cells were not completely resistant to amino-acid starvation, as S6K activity was reduced by 50% in this cell line after 2 h of starvation. This is perhaps a result of the fact that the Eker allele is not a molecular null mutation. In contrast, when the requirement of Tsc1 in amino acid signalling was examined using CACL-1-111, a Tsc1^-/- cell line derived from a Tsc1 knockout mice (Kobayashi, 2001), this cell line was completely resistant to amino-acid starvation, despite being sensitive to rapamycin. Thus, these results strongly implicate Tsc1-Tsc2 in amino acid signalling (Gao, 2002).

It is proposed that the Tsc1 and Tsc2 tumour suppressors are negative regulators of the amino acid-TOR pathway that normally couples the availability of amino acids to protein synthesis. Genetic and biochemical studies are consistent with three possible models of Tsc1-Tsc2 function. In models 1 and 2, which this study favour, Tsc1-Tsc2 could either function as an obligatory component between amino acids and TOR in a linear amino acid sensing pathway, or negatively regulate the ability of TOR to sense amino-acid levels. These two models are based on the assumption that amino acids signal through TOR, a notion that is supported by peripheral evidence in mammalian, but remains unproven. However, the possibility cannot be excluded that Tsc1-Tsc2 functions in parallel to TOR to control S6K (model 3). An implication of this model, given the resistance of Tsc1-Tsc2 mutant cells to amino-acid starvation, is that there are at least two pathways by which amino acids signal to S6K: one involving TOR and the other parallel to TOR, but antagonized by Tsc1-Tsc2. Further investigations will be required to distinguish between these possibilities. It is also worth noting that loss of Tsc1-Tsc2 results in increased phosphorylation of both S6K and 4E-BP. Thus, the protein target of Tsc1-Tsc2 must regulate both S6K and 4E-BP. This protein could be TOR or an activator of TOR (models 1 and 2). Alternatively, it could be an unidentified common activator of both S6K and 4E-BP (Gao, 2002).

These studies have provide the first example of tumour suppressors that are involved in nutrient sensing. Although many tumour suppressor genes are involved in aspects of cell signalling, such as growth factor receptor signalling and various cell cycle checkpoints, Tsc1-Tsc2 can be viewed as a component of an analogous 'nutrient checkpoint', which senses amino-acid availability. Aberrant regulation of this important signalling pathway can result in defects in the ability of a cell to respond to nutrient levels and cause neoplasm. The results further suggest that rapamycin, or its derivatives that target TOR, may be used to potential therapeutic advantage against tuberous sclerosis syndrome (Gao, 2002).

Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size

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).

TSC is a common disease with severe clinical consequences. This study presents the characterization of a TSC1 homolog in Drosophila. Since homologs of TSC1 and TSC2 are not found in yeast or C. elegans, Drosophila is an invaluable model for dissecting the in vivo functions of TSC1 and TSC2 (Potter, 2001).

The most dramatic phenotype of Tsc1 is an alteration in cell size. Mutation of Tsc1 in adult eye or wing structures result in an average 3-fold increase in cell size in a cell autonomous fashion. The observation that both 2C and 4C Tsc1 mutant cells in third instar wing and eye discs are increased in size, and that the mutant cells in the anterior proliferating region of the eye disc are larger than wild-type, suggests that an increase in size occurs in proliferation cells during all stages of the cell cycle. Since Tsc1 mutant cells in third instar discs are not as large as the mutant cells in pupae and adults, it is possible that this difference reflects that cells committed to terminal differentiation have more time to grow. Flow cytometry analysis indicated that the increase in cell size was not due to endoreplication. In addition, overexpression of Tsc1, along with its functional partner Tsc2, results in dramatic decreases in cell size in the eye and the wing. Despite the alterations in cell size, the differentiation of adult structures is largely unaffected (Potter, 2001).

Alterations of Tsc1 function also lead to changes in cell number. Co-overexpression of Tsc1 and Tsc2 in the eye or wing leads to a 15% decrease in cell numbers. Immunohistochemistry analysis shows that loss of Tsc1 function allows eye imaginal disc cells that should be arrested at G₀ to enter the cell cycle. Futhermore, the Tsc1 mutant wing imaginal disc cell population analyzed by flow cytometry exhibits a significant decrease in the percentage of cells at G₁/G₀. Similarly, immortalized fibroblasts derived from the Eker TSC2 mutant rat show a decrease in cells at G₁/G₀. It is suggestd that Tsc1 affects the regulation of G₁/G₀. The mechanism by which Tsc1 regulates cell proliferation is unknown (Potter, 2001).

The molecular mechanisms that control organ size are not fully understood. Genetic screens in Drosophila, however, have identified three classes of mutations that affect organ size. Mutations in Drosophila tumor suppressors, such as lats, cause dramatic overproliferation, which results in tumorous growth of mutant cells in mosaic animals and enlarged organs in homozygous mutants. Mutations in the second class, such as slimb, cause duplicated outgrowths in mosaic animals and altered organ size by affecting signals that regulate pattern formation. Mutations in the third class, such as dPTEN, cause overgrowth of mutant clones in mosaic animals, but do not disrupt normal patterning. While the first two classes of mutations affect organ size mainly by increasing cell numbers, mutations of the third class affect organ size largely by affecting cell size (Potter, 2001).

Tsc1 is another gene identified in mosaic screens that affect organ size. Eyes consisting primarily of Tsc1 mutant cells are increased in size by 2.5 to 3 times. Surprisingly, these eyes contain relatively normal numbers of ommatidia in comparison to controls. Analysis of late pupal eye discs revealed that 40% of the ommatidia have, on average, 1.5 extra cells. This translates to a 1% increase in eye size due to the increase in cell number. Thus, the dramatic increase in size of the Tsc1 mutant eye is largely contributed by the increase in cell size. Similarly, cell size reduction contributes ~86% of the overall decrease in wing size in wings that co-overexpress Tsc1 and Tsc2, while cell number reduction contributes only ~14% to the decrease in wing size. Therefore, Tsc1 is a member of the class III mutations and affects organ size primarily by altering the size of the cell. Since the Tsc genes can affect cell and organ size in multiple tissues, it might represent a global regulator of growth in Drosophila (Potter, 2001).

The results provide in vivo evidence that Tsc1 and Tsc2 function together as a unit. Similar to studies with mammalian TSC proteins, it was found that Tsc1 and Tsc2 bind in vitro. Furthermore, overexpression of Tsc1 or Tsc2 alone was found to have no effect, whereas co-overexpression dramatically affects cell size, cell proliferation, and organ size. In addition, in both humans and flies, mutations of the two TSC genes give rise to phenotypes that are indistinguishable, strongly suggesting that removal of either gene equally affects an identical function. It is proposed that the binding of TSC1 and TSC2 results in a functional unit. Interestingly, all known mutations affecting TSC1 function are predicted to truncate the protein. It is suggested that individual missense mutations in TSC1 would unlikely eliminate the binding of TSC1 to TSC2. How the binding of TSC1 and TSC2 might result in an activity that neither alone contains is unclear. Perhaps a TSC1/TSC2 complex allows for proteins bound to TSC1 to interact with proteins bound to TSC2. Alternatively, the binding of TSC1 and TSC2 might alter their conformations, and allow for the interactions of downstream players. A number of proteins (i.e., ERM-family members, rap1) have been found to bind either TSC1 or TSC2. It would thus be interesting to determine the functional relationship between these proteins and the TSC1/TSC2 complex (Potter, 2001).

Tsc1 is a potent regulator of cell growth, cell proliferation, and organ size in Drosophila. Many recent studies have shown that components of the insulin signaling pathway also affect these same properties. For example, inactivation of dPTEN, a negative regulator of insulin signaling, or overexpression of dinr, lead to an increase in cell size, cell number, and organ size. In contrast, overexpression of dPTEN results in opposite phenotypes, which are akin to the effects caused by co-overexpression of Tsc1 and Tsc2, or inactivation of the positive components of the insulin pathway (dinr. Genetic epistasis experiments show that cells that are double mutant for Drosophila Tsc (Tsc1 or Tsc2) and dAkt (or dinr or ovexpression of dPTEN) display the Tsc1/Tsc2 inactivation phenotype, and cells that overexpress Tsc1, Tsc2, and dinr exhibit the Tsc1/Tsc2 co-overexpression phenotype. Thus, Tsc1 and Tsc2 are genetically epistatic to dAkt and to upstream components of the insulin pathway (Potter, 2001).

The dS6k gene has been shown to act as a downstream component in the insulin pathway in the regulation of cell growth. This study found that cells mutant for both Drosophila Tsc (Tsc1 or Tsc2) and dS6k display the dS6k small cell mutant phenotype, and overexpression of dS6k suppresses the Tsc1/Tsc2 co-overexpression phenotype. These data indicate that dS6k is genetically epistatic to Tsc1 and Tsc2 (Potter, 2001).

These genetic data are consistent with a model in which Tsc1 and Tsc2 function together downstream of dAkt and upstream of dS6k in the insulin pathway (See Genetic Models for the Functions of Tsc1 and Tsc2). Alternatively, Tsc1 and Tsc2 could function in a parallel pathway or in a pathway that converges with insulin signaling. Currently, these possibilities can not be excluded. Recently, mutations of the Drosophila TOR and lilliputian (lilli) genes have been found to affect cell size. However, unlike Tsc1 and Tsc2, lilli and dTOR have additional phenotypes that differ from alterations in components of the insulin pathway. Furthermore, double mutant analysis between lilli and dPTEN could not establish an epistatic relationship. Consequently, lilli and dTOR have been proposed to function in parallel or converged pathways (Potter, 2001).

The major clinical symptom of TSC is the development of hamartomas. Interestingly, hamartomas often share many of the characteristics associated with Tsc1 mutant clones: enlarged cells, overproliferation, and normal differentiation. Hamartomas might therefore result from deregulation of the insulin signaling pathway. In support of this hypothesis, Cowden syndrome, which is caused by germline mutations in PTEN, is also characterized by the presence of hamartomas in multiple organs. The possibility that TSC1 and TSC2 function in the insulin pathway emphasizes the importance of this pathway in the development of cancers and other diseases. These results further suggest that TSC-related lesions may be treated by targeting S6K or other downstream components of the insulin signaling pathway (Potter, 2001).

TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth

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 mechanisms of how body and organ size are regulated are largely unknown. Recent genetic studies in Drosophila suggest that the insulin pathway may coordinately control both cell growth and cell proliferation and in turn regulate organ size. This study has provided evidence that theTSC tumor suppressor genes also play an essential role in the control of cell size and organ size. Although an increase in cell size has been observed in human tumors carrying TSC1 orTSC2 mutations, the underlying mechanisms are not clear. The current results suggest that theTSC genes and the insulin pathway act antagonistically in the control of cellular growth. Loss of TSC genes resulted in a cell-size phenotype that is almost identical to that of PTEN. Similarly, co-overexpression of the TSC genes reduces cell size as PTEN overexpression. The results from double-mutant analyses suggest that the TSC genes act in a pathway parallel to the insulin pathway, and the TSC pathway converges on the insulin pathway downstream from Akt. Several proteins are known to function directly downstream from Akt, or to converge on the insulin pathway downstream from Akt. These include S6K, 4E-BP, and Ser/Thr kinase TOR. The genetic analysis is consistent with the TSC pathway regulating any of these candidate proteins. Alternatively, the TSC genes may regulate unknown regulators of cell growth. It is not possible distinguish between these models at present (Gao, 2001).

The most convincing evidence for a functional link between theTSC genes and insulin signaling came 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).

An important challenge in the future is to understand the molecular mechanism by which the TSC tumor suppressors regulate the insulin pathway. The predicted structures of the TSC1 (containing coiled-coil domains) and the TSC2 protein (containing a GAP domain) offer clues in that regard. The TSC2 protein has been shown to possess GAP activity toward two small GTPases, Rab5 and Rap1. Rab5 is a rate-limiting component of the endocytic pathway. It has been speculated that loss of the TSC tumor suppressors could lead to missorting of internalized growth factor receptors or other signal-mediated membrane-bound molecules that would otherwise undergo lysosomal degradation, thus leading to a constitutive activation of certain growth-promoting pathways. The in vivo function of Rap1 is largely unknown, and Rap1 can function both as positive and negative regulator of cell proliferation under different conditions. Further studies of the Drosophila TSC genes may provide insights into the relative importance of Rab5GAP and Rap1GAP activities in growth suppression (Gao, 2001).

The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder affecting 1 in 5800 individuals. TSC occurs in multiple organs, including the brain, eyes, skin, kidney, heart, lungs, and skeleton, and is characterized by the presence of benign tumor cells termed hamartomas. Hamartomas are a mass of disorganized but differentiated cells indigenous to the site. TSC hamartomas rarely progress to malignancy, but brain hamartomas frequently cause epilepsy, mental retardation, autism, or attention deficit-hyperactive disorder. One of the notable features of TSC hamartomas is the presence of giant cells in the tumors. Linkage studies in families with TSC have established two TSC loci, TSC1 and TSC2, each accounting for approximately 50% of cases. The TSC1 gene encodes a novel protein, hamartin, that contains a single transmembrane domain and a large cytoplasmic tail with coiled-coil domains (van Slegtenhorst, 1997). The TSC2 gene, the homolog of Drosophila Gigas and the subject of this overview, encodes a novel protein, tuberin, that contains a region of homology to the GTPase-activating protein (GAP) for the small-molecular-weight GTPase Rap1. Clones of gigas mutant cells induced in imaginal discs differentiate normally to produce adult structures. However, the cells in these clones are enlarged (gigas means 'giant' in Latin) and repeat S phase without entering M phase. This result suggests that the TSC disorder may result from an underlying defect in cell cycle control (Ito, 1999 and references). Recent studies have focused on the role of the TSC genes as modifiers of the insulin pathway (Tapon, 2001, Potter, 2001 and Gao, 2001). In particular, it is unlikely that deficiencies in TSC gene function results in alterations in cell polyploidy (Ito, 1999), but instead the growth and size defects may arise through defective signaling in the insulin pathway (Tapon, 2001, Potter, 2001 and Gao, 2001).

Mutations have been characterized in both Tsc1 and Tsc2/gigas genes of Drosophila. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions (Tapon, 2001).

The disease phenotype in humans and observations in Drosophila suggest that all the functions of TSC1 and TSC2 are mediated by a complex of the two proteins, and that neither protein has additional functions. Moreover, combined overexpression of both Tsc1 and Tsc2 readily elicits a phenotype in a variety of tissues, while overexpression of either protein alone is insufficient. This argues that most, if not all, of the endogenous Tsc1 and Tsc2 is sequestered in the complex and is unavailable to complex with exogenously supplied protein. Cells mutant for either Tsc1 or Tsc2 are larger than wild-type cells and show a marginal decrease in their division time. Thus, the rate of mass accumulation (growth) in Tsc1 and Tsc2 mutant cells must be greater than that of wild-type cells to allow them to be larger than their neighbors despite a similar division time. Hence, the primary alteration in the mutants may be an increase in the rate of cellular growth. Conversely, when both Tsc1 and Tsc2 are overexpressed, the growth rate of the tissue is reduced. The cells are smaller and take longer to divide than wild-type cells. The increase in duration of the cell cycle may represent an attempt to maintain a near normal cell size in a situation where the rate of growth is reduced. An alternate possibility is that Tsc1 and Tsc2 negatively regulate both cell growth and cell cycle progression independently (Tapon, 2001).

The increased growth rate of mutant tissue is apparent in both cycling and postmitotic cells. In cycling populations such as the wing disc and in the eye disc anterior to the morphogenetic furrow, mutant clones are larger than the corresponding wild-type twin spots. The difference in size between the clone and the twin-spot becomes more exaggerated after the cells have stopped dividing, resulting in a greatly increased representation of mutant tissue in the adult (Tapon, 2001).

A major pathway that regulates growth in Drosophila is the pathway that links signaling via the insulin receptor to phosphorylation of the ribosomal protein S6. This is thought to lead to increased ribosome biosynthesis and mass accumulation. Thus, loss-of-function mutations in the insulin receptor (inr), the insulin receptor substrate (chico), and in genes encoding the downstream signaling molecules PI3K, Akt (Dakt), and S6 kinase (dS6K), all reduce cell and organ size. Conversely, overexpression of PI3K or loss-of-function mutations in the insulin pathway antagonist PTEN (dPTEN) lead to increased cell and tissue growth. In addition, Ras1, possibly acting via dmyc, can also promote cell growth in response to extracellular growth factors. Increased activity of Cyclin D in a complex with cdk4 has also been shown to be a potent stimulus for growth in both dividing and postmitotic cells (Tapon, 2001 and references therein).

The enhanced growth observed in the Tsc1 or Tsc2 mutants most resembles the results of inactivating PTEN or increasing Ras1 or dmyc activity. In each of these situations, there is a reduction in the length of the G1 phase. In contrast, increased growth driven by Cyclin D/cdk4 does not alter the distribution of cells in different phases of the cell cycle. The effects of the combined overexpression of Tsc1 and Tsc2 displays genetic interactions with multiple pathways. The phenotype is influenced by alterations in the levels of dS6K, PTEN, Ras1, dmyc, cyclin D, and cdk4. Thus, Tsc1 and Tsc2 may function downstream of the point of convergence of these pathways. Alternatively, Tsc1 and Tsc2 may primarily antagonize one of these pathways, but this effect could be overcome by increasing the activity of one of the others (Tapon, 2001).

Imaginal discs containing large mutant clones of either Tsc1 or Tsc2 are significantly larger than wild-type imaginal discs but are patterned relatively normally. Mutations in several cell size regulators such as E2F or Rbf have been shown to affect the size of individual cells but do not alter the final size of the organ. The mechanisms that define organ size are poorly understood. The complex of Tsc1 and Tsc2 restricts the growth of organs in vivo. Screens for genes that interact with Tsc1 and Tsc2 are likely to identify additional regulators of organ size (Tapon, 2001).

Tsc1 and Tsc2 may modulate the cell cycle via changes in cyclin levels. In Tsc1 and Tsc2 mutant clones, the levels of both Cyclin E and Cyclin A are elevated. Cell growth driven by dmyc or Target of rapamycin (dTor) elevates Cyclin E levels. It has been postulated that Cyclin E may function as a 'growth sensor' in a manner analogous to CLN3 in yeast and that the translation of Cyclin E is more efficient in cells that have an increased rate of growth. The increased levels of Cyclin E may be responsible for the shortening of G1 in Tsc1 and Tsc2 mutants. It is unclear why Cyclin A and Cyclin B are also elevated in mutant cells. Cyclin A is normally expressed at high levels in G2. In Tsc1 or Tsc2 mutants, Cyclin A levels are elevated in the post-mitotic cells of the eye disc that are clearly not arrested in G2. Thus it seems likely that the increased growth in mutant cells may also lead to increased levels of mitotic cyclins. Alternatively Tsc1 and Tsc2 may function in a pathway that negatively regulates cyclin levels (Tapon, 2001).

While the increased levels of cyclins are likely to be a response to the increased growth rate of mutant cells, the possibility that they are in some way responsible for the increased growth rate cannot be excluded. In Drosophila, the Cyclin D/cdk4 complex serves to promote growth. In such a scenario, the loss of Tsc1 or Tsc2 gene function may lead to elevated levels of cyclins leading to increased growth and proliferation. Surprisingly, increased expression of Cyclin E, which is thought to primarily promote S-phase entry and not growth, is also able to suppress the phenotype induced by overexpression of Tsc1 and Tsc2. This might reflect the existence of feedback loops where Cyclin E might downregulate the levels or activity of the Tsc1/Tsc2 complex. Alternatively, in some circumstances, Cyclin E might assume some of the functions of the growth promoting Cyclin D. Indeed, in mammalian cells, cyclin E has been shown to fully compensate for the loss of cyclin D1 (Tapon, 2001).

Tsc mutant cells fail to maintain a developmentally induced G1 arrest posterior to the second mitotic wave in third instar eye imaginal discs. The establishment of this G1 arrest requires a downregulation of Cyclin E and Cyclin A expression. However, the transient cell cycle arrest in the morphogenetic furrow occurs normally in Tsc1 and Tsc2, suggesting that it is the maintenance of G1 arrest that is perturbed rather than its initial establishment. Postmitotic cells continue to grow abnormally in Tsc1 and Tsc2 mutants and express elevated levels of Cyclin E and Cyclin A. A likely model is that inappropriate and continued growth in postmitotic cells leads to an accumulation of Cyclin E and the mitotic cyclins. This would eventually force cells to overcome a developmentally regulated cell cycle arrest and to reenter the cell cycle. Indeed, many of the lesions in patients with TSC occur in organs that consist predominantly of postmitotic cells such as the heart and brain. A successful therapeutic strategy in tuberous sclerosis is likely to be one that can curtail the inappropriate cell growth (Tapon, 2001).

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).

The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1

The cochaperone BAG3 (Starvin in Drosophila) is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. This study demonstrates that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 (see Drosophila TSC1) that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain (Kathage, 2017).

Tsc1 mutant neural stem/progenitor cells exhibit migration deficits and give rise to subependymal lesions in the lateral ventricle.

Subependymal nodules (SENs) and subependymal giant cell astrocytomas (SEGAs) are common brain lesions found in patients with tuberous sclerosis complex (TSC). These brain lesions present a mixed glioneuronal phenotype and have been hypothesized to originate from neural stem cells. However, this hypothesis has not been tested empirically. This study reports that loss of Tsc1 in mouse subventricular zone (SVZ) neural stem/progenitor cells (NSPCs) results in formation of SEN- and SEGA-like structural abnormalities in the lateral ventricle, the consequence of abnormal migration of NSPCs following Tsc1 loss (Zhou, 2011).

Considering the many TSC-associated brain lesions, the observation that loss of Tsc1 in the NSPC population alone is sufficient to cause aberrant neuroblast migration may have larger implications. Migration deficits following Tsc1 loss may be a general underlying mechanism for other TSC-related brain lesions as well. For example, cortical tubers, the most common TSC-associated brain lesion, are characterized by disrupted cortical lamination and the presence of aberrant glia and neurons within the tuber, possibly due to a migration problem of the NSCs during formation of the cortical layers. This idea is supported by a recent study in which inducing Tsc1 loss in a small subset of cortical progenitor cells by in utero electroporation at embryonic day 15 (E15) resulted in tuber-like structure formation (Feliciano, 2011). However, while this study has demonstrated that Tsc1 loss in ependymal cells is not required for the Tsc1-null NSPC migration deficits, the possibility is not excluded that loss of Tsc1 in ependymal cells can facilitate tumor formation or contribute to the development of hydrocephalus in this model (Zhou, 2011).

While the tumors recapitulate several key features of human SEGAs, pleomorphic multinucleated tumor cells that are frequently seen in human SEGAs were not seen. It is noted that mouse models of cortical tubers also possess some, but not all, histological features of tubers. This may reflect differences between species, or it may be that some histological features take a longer time to develop, and in the mouse model, only the initial stages of SEGA development were captured (Zhou, 2011).

One interesting question is why SEGAs preferentially develop near the foramen of Monro. In the mouse model, nodular structures that developed on the lateral ventricle wall did not become very large and tended to 'fall' into the ventricle, whereas similar cell masses that formed near the IF grew bigger and developed into small tumors. This ventricular region might be advantageous for tumor development due to its stem cell-rich environment and the narrow ventricular space, which could facilitate formation of initial cell masses and provide a supportive growth environment. It is possible that multiple cell masses develop simultaneously and then merge into one large mass. This is supported by the observation of individual cell masses in this area. This idea has also been proposed in human SEGA development; it is believed that SEGAs originate from SENs near the foramen of Monro (Zhou, 2011).

In summary, this study has showm that Tsc1 loss specifically in NSPCs can drive the development of SEN- and SEGA-like structures in the ventricles, due to aberrant aggregation and migration of SVZ NSPCs following Tsc1 loss. This study offers direct genetic evidence that the cells of origin of human SENs and SEGAs are the NSPCs, thus providing new insight into TSC1/2 function in the nervous system (Zhou, 2011).

Search PubMed for articles about Drosophila Tsc1

Amcheslavsky, A., Ito, N., Jiang, J. and Ip, Y. T. (2011). Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells. J. Cell Biol. 193(4): 695-710. PubMed ID: 21555458

Bateman, J. M. and McNeill, H. (2004). Temporal control of differentiation by the Insulin receptor/Tor pathway in Drosophila. Cell 119: 87-96. PubMed ID: 15454083

Benmimoun, B., Polesello, C., Waltzer, L. and Haenlin, M. (2012). Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila. Development 139: 1713-1717. PubMed ID: 22510984

Canal, I., Acebes, A. and Ferrus, A. (1998). Single neuron mosaics of the Drosophila gigas mutant project beyond normal targets and modify behavior. J. Neurosci.18(3): 999-1008. PubMed ID: 9437021

Chalhoub, N., Kozma, S. C. and Baker, S. J. (2006). S6k1 is not required for Pten-deficient neuronal hypertrophy. Brain Res. 1100: 32-41. PubMed ID: 16777079

Chell, J. M. and Brand, A. H. (2010). Nutrition-responsive glia control exit of neural stem cells from quiescence. Cell 143: 1161-1173. PubMed ID: 21183078

Chong-Kopera H, Inoki K, Li Y, Zhu T, Garcia-Gonzalo FR, Rosa JL, Guan KL. TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase. J. Biol. Chem. 281(13): 8313-6. PubMed ID: 16464865

Feliciano, D. M., et al. (2011). Single-cell Tsc1 knockout during corticogenesis generates tuber-like lesions and reduces seizure threshold in mice. J. Clin. Invest. 121: 1596-1607. PubMed ID: 21403402

Gao, X. and Pan, D. (2001). TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth. Genes Dev. 15: 1383-1392. PubMed ID: 11390358

Gao, X., et al. (2002). Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat. Cell Biol. 4(9): 699-704. PubMed ID: 12172555

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

Hsu, Y.-C., Chern, J. J., Cai, Y., Liu, M. and Choi, K.-W. (2007). Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase. Nature 445: 785-788. PubMed ID: 17301792

Ito, N. and Rubin, G. M. (1999). gigas, a Drosophila homolog of tuberous sclerosis gene product-2, regulates the cell cycle. Cell 96(4): 529-39. PubMed ID: 10052455

Kapahi, K., et al. (2004). Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr. Biol. 14: 885-890. PubMed ID: 15186745

Kathage, B., Gehlert, S., Ulbricht, A., Ludecke, L., Tapia, V. E., Orfanos, Z., Wenzel, D., Bloch, W., Volkmer, R., Fleischmann, B. K., Furst, D. O. and Hohfeld, J. (2017). The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1. Biochim Biophys Acta Mol Cell Res 1864(1): 62-75. PubMed ID: 27756573

Knox, S., et al. (2007). Mechanisms of TSC-mediated control of synapse assembly and axon guidance. PLoS ONE 2(4): e375. PubMed ID: 17440611

Kobayashi, T. et al. (2001). A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice Proc. Natl Acad. Sci. 98: 8762-8767. PubMed ID: 11438694

Layalle, S., Arquier, N., Léopold, P. (2008). The TOR pathway couples nutrition and developmental timing in Drosophila. Dev. Cell 15(4): 568-77. PubMed ID: 18854141

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

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

Martin-Pena, A., et al. (2006). Age-independent synaptogenesis by phosphoinositide 3 kinase. J Neurosci 26: 10199-10208. PubMed ID: 17021175

Murthy, V., et al. (2004). Pam and its ortholog highwire interact with and may negatively regulate the TSC1.TSC2 complex. J Biol Chem 279: 1351-1358. PubMed ID: 14559897

Potter, C. J., Huang, H. and Xu, T. (2001). Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 105: 357-368. PubMed ID: 11348592

Sousa-Nunes, R., Yee, L. L. and Gould, A. P. (2011). Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila. Nature 471(7339): 508-12. PubMed ID: 21346761

Sun, P., Quan, Z., Zhang, B., Wu, T. and Xi, R. (2010). TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation. Development 137(15): 2461-9. PubMed ID: 20573703

Tapon, N., et al. (2001). The Drosophila Tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell 105: 345-355. PubMed ID: 11348591

Tavazoie, S. F., et al. (2005). Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and Tsc2. Nat. Neurosci. 8: 1727-1734. PubMed ID: 16286931

Thomas, G. (2002). The S6 kinase signaling pathway in the control of development and growth. Biol. Res. 35(2): 305-13. PubMed ID: 12415748

Tokusumi, Y., Tokusumi, T., Shoue, D. A., Schulz, R. A. (2012). Gene regulatory networks controlling hematopoietic progenitor niche cell production and differentiation in the Drosophila lymph gland. PLoS One 7(7):e41604. PubMed Citation: 22911822

van Slegtenhorst, M., de Hoogt, R., Hermans, C., Nellist, M., Janssen, B., Verhoef, S., Lindhout, D., van den Ouwerland, A., Halley, D. and Young, J. et al. (1997). Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277: 805-808. PubMed ID: 9242607

Wullschleger, S., Loewith, R. and Hall, M. N. (2006). TOR signaling in growth and metabolism. Cell 124: 471-484. PubMed ID: 16469695

Zhang, Y., et al. (2003). Rheb is a direct target of the tuberous sclerosis tumor suppressor proteins. Nat. Cell Biol. 5(6): 578-81. PubMed ID: 12771962

Zhou, J., et al. (2011). Tsc1 mutant neural stem/progenitor cells exhibit migration deficits and give rise to subependymal lesions in the lateral ventricle. Genes Dev. 25(15): 1595-600. PubMed ID: 21828270

date revised: 10 December 2020

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