Phosphotidylinositol 3 kinase 92E


Expression pattern of class I phosphoinositide 3-kinase and distribution of its product

The class I phosphoinositide 3-kinase (PI3K) can be activated by a large variety of extracellular stimuli and is responsible for generating phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) from phosphatidylinositol-4,5-bisphosphate at the plasma membrane. The expression pattern of the class I PI3K and distribution of PI(3,4,5)P3, visualized by its specific binding protein, GRP1-PH, were examined during Drosophila embryogenesis. The RNA of Pi3K21B, encoding the Drosophila p60 regulatory subunit of the class I PI3Ks, is expressed maternally and expressed primarily in pole cells after cellularization until completion of germ band elongation. The RNA of Pi3K92E, encoding the Drosophila p110 catalytic subunit of the class I PI3Ks, is also expressed maternally. During gastrulation, its transcript level becomes lower and is slightly enriched in invaginating cells. Both Pi3K21B and Pi3K92E are expressed ubiquitously after germ band elongation and persist during germ band shortening. PI(3,4,5)P3 is distributed at the apical region of the invaginating cells during gastrulation. These findings suggest a possible involvement of class I PI3K and PI(3,4,5)P3 in the regulation of invagination during gastrulation (Xi, 2014).

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).

Nutrition-responsive glia control exit of neural stem cells from quiescence

The systemic regulation of stem cells ensures that they meet the needs of the organism during growth and in response to injury. A key point of regulation is the decision between quiescence and proliferation. During development, Drosophila neural stem cells (neuroblasts) transit through a period of quiescence separating distinct embryonic and postembryonic phases of proliferation. It is known that neuroblasts exit quiescence via a hitherto unknown pathway in response to a nutrition-dependent signal from the fat body. This study has identified a population of glial cells that produce insulin/IGF-like peptides in response to nutrition, and shows that the insulin/IGF receptor pathway is necessary for neuroblasts to exit quiescence. The forced expression of insulin/IGF-like peptides in glia, or activation of PI3K/Akt signaling in neuroblasts, can drive neuroblast growth and proliferation in the absence of dietary protein and thus uncouple neuroblasts from systemic control (Chell, 2010).

A transcriptome analysis comparing VNCs from newly hatched larvae and VNCs from larvae at the end of the first instar suggested that the expression of dILP6 and dILP2 increases in the VNC during neuroblast reactivation. The seven dILPs are expressed in distinct spatiotemporal patterns during development. dILP6 is reported to be expressed in the larval gut and the pupal fat body , whereas dILP2 is known to be expressed in the IPC neurons of the brain (along with dilps 1, 3, and 5). To determine whether dILP6 is also expressed in the CNS, a dilp6-GAL4 line was generated. dilp6-GAL4 drives expression in a subset of the surface glia that wraps the CNS. Strong expression was evident by mid first instar and was maintained throughout neuroblast reactivation. The expression of dILP2 was assayed by immunohistochemistry; it too was expressed in the same surface glial population. The glial cells labeled by dilp6-GAL4 are located above the neuroblasts and underneath the surrounding basement membrane. They are stellate in appearance, with several processes radiating from the central cell body. Thus, dILPs, expressed by glial cells, are ideally positioned to activate the dInR pathway in neuroblasts during reactivation (Chell, 2010).

Drosophila Akt is a key transducer of increased PIP3 levels, such as those seen in response to dInR/PI3K activation. Following recruitment to the cell membrane, Akt is activated by PDK1-mediated phosphorylation. This study found that, when PI3K activity was increased in neuroblasts by expression of dp110CAAX (a membrane-targeted, constitutively active, version of the PI3K catalytic subunit), the levels of phosphorylated Akt (pAkt) were concomitantly increased. To test whether Akt activation is sufficient for the exit from quiescence, a membrane-targeted form of Akt (myr-Akt) was expressed in neuroblasts of larvae reared on a sucrose-only diet. myr-Akt expression was sufficient to drive both growth and cell-cycle re-entry (as evidenced by extensive pH3 labeling) in quiescent neuroblasts in the absence of the nutritional stimulus. Indeed, expression of myr-Akt was more potent than dp110CAAX, as all grh-GAL4- positive neuroblasts reactivated. The difference in the number of neuroblasts that reactivated in response to dp110CAAX (4%–12%) and myr-Akt (100%) may reflect a differential sensitivity to negative feedback regulation in the pathway. Myr-Akt may escape negative control more readily than wild-type Akt that has been activated by dp110CAAX (Chell, 2010).

Once neuroblast reactivation has been ectopically triggered by either PI3K or Akt, then neuroblast proliferation occurs at approximately the same rate. When reactivated neuroblasts were assayed at 24 hr, they had generated on average six or seven daughter cells under either condition. For dp110CAAX, the daughter cells of 29 reactivated neuroblasts from 10 tVNCs were counted; on average, each neuroblast had 6.76 daughter cells. For myr-Akt, the daughter cells of 40 reactivated neuroblasts from four tVNCs were counted; on average, each neuroblast had 6.65 daughter cells. Thus, dInR/PI3K appear to act via their canonical downstream pathway, and when activated in neuroblasts, this pathway is sufficient for reactivation (Chell, 2010).

Drosophila neuroblasts in the central brain and thoracic ventral nerve cord (tVNC) are quiescent for 24 hours between their embryonic and larval phases of proliferation. Quiescent neuroblasts are easily identifiable and are amenable to genetic manipulation, making them a potentially powerful model with which to study the transition between quiescence and proliferation. However, the mechanisms regulating the exit from quiescence, either intrinsic or extrinsic, are not well established. Genetic studies found that Drosophila FGF, in concert with Drosophila Perlecan, promotes the neuroblast transition from quiescence to proliferation, but this effect is indirect (Barrett, 2008). Exit from quiescence is physiologically coupled to larval growth and development via a nutritional stimulus (Britton, 1998). The Drosophila fat body performs many of the storage and endocrine functions of the vertebrate liver and acts as a sensor, coupling nutritional state to organismal growth. In response to dietary amino acids, the fat body secretes a mitogen that acts on the CNS to bring about neuroblast proliferation (Britton, 1998). This fat body-derived mitogen (FBDM) initiates cell growth in quiescent neuroblasts and promotes (or at least permits) cell-cycle re-entry (Britton, 1998). Yet the identity of the FBDM, the cell type upon which it acts, and the downstream pathway activated in neuroblasts have remained unknown (Chell, 2010).

Neuroblast entry into quiescence is governed intrinsically by the same transcription factor cascade that controls neuroblast temporal identity. This study has identified a population of surface glial cells that respond to the nutrition-dependent stimulus by expressing dILPs, and showns that the dInR/PI3K pathway is required by neuroblasts to exit quiescence in response to nutrition. Forced expression of dILPs in glia or activation of PI3K/Akt signaling in neuroblasts can drive neuroblast growth and proliferation in the absence of dietary protein and thus uncouple neuroblast reactivation from systemic nutritional control (Chell, 2010).

Cell growth and division are not strictly coupled in neuroblasts. In Drosophila Perlecan (dPerlecan) loss-of-function mutants, the majority of neuroblasts appear to increase in size but then remain G1 arrested. This suggested that a dedicated mitogen might exist to promote cell-cycle progression. Drosophila Activin-like peptides (ALPs; Zhu, 2008) are required for normal levels of neuroblast division in the larval brain and appear to be one such dedicated mitogen (Chell, 2010).

Perlecan is expressed by glia and forms part of the basement membrane that enwraps the CNS. Perlecan was proposed to modulate Drosophila FGF [Branchless (Bnl)], allowing it to act as a mitogen for neuroblasts. However, it now appears that the action of Bnl is indirect via a still to be identified cell type (Barrett, 2008). One possibility is that Bnl acts on glia to modulate the expression of other proteins, such as dILPs or ALPs, which then in turn act on neuroblasts directly. This study shows that expression of dILPs by glia leads to neuroblast reactivation in the absence of dietary protein; however, the number of mitoses falls short of that seen under normal dietary conditions. This could be explained by the absence of another nutritionally dependent mitogen. It will be of interest to see whether the glial expression of ALPs, like that of dILPs, relies on dietary protein (Chell, 2010).

In the larval CNS, neuroblasts and their progeny are completely surrounded by glial cell processes. If the interaction between neuroblasts and surrounding glia is disrupted by expression of a dominant-negative form of DE-cadherin, the mitotic activity of neuroblasts is severely reduced (Dumstrei, 2003). In the mammalian brain, glial cells are involved in a wide variety of processes, including axon guidance, synapse formation, and neuronal specification. Glial cells, with the extracellular matrix and vasculature, also make up the adult neural stem cell niche. Astrocytes have been shown to promote neural stem cell proliferation in culture and can express proproliferative factors such as FGF-2 and IGF-I. Thus, astrocytes are thought to be a key component of the niches that dynamically regulate neural stem cell proliferation in the adult brain (Chell, 2010).

This study has shown that Drosophila surface glia can transduce systemic signals and, by expressing dILP2 and dILP6, control neuroblast exit from quiescence. Glial cells also express dPerlecan and ana and are the source of the Activin-like peptides that have a direct mitogenic effect on neuroblasts. Thus, much like mammalian glial cells, Drosophila glial cells perform a number of the functions that define a niche and control the proliferation of neural stem cells (Chell, 2010).

Recent results suggest a role for IGF-1 in the control of neural stem cell division (Mairet-Coello et al., 2009). IGF-1 injection into rat embryonic brain results in a 28% increase in DNA content postnatally as a consequence of increased DNA synthesis and entry into S phase. Conversely, DNA synthesis and entry into S phase are decreased when the PI3K/Akt pathway is blocked. Furthermore, the loss of PTEN, the tumor suppressor and PI3K antagonist, enhances the exit from G0 of neural stem cells cultured from mouse embryonic cortex. It was suggested that a concomitant increase in cell size may push the cells to enter G1 (Chell, 2010).

This study shows, in vivo, that glial expression of insulin-like peptides activates the dInR/PI3K/Akt pathway in Drosophila neural stem cells and is responsible for their exit from quiescence. This pathway promotes cell growth and the transition from G0 to G1 and is also sufficient to promote G1-S and mitosis. Given that IGF-1 and the PI3K/Akt pathway can promote cell-cycle progression in vertebrate neural stem cells, this same pathway may regulate vertebrate neural stem cell reactivation in the same way as has been shown in this study for Drosophila (Chell, 2010).

The identity of the proposed FBDM, secreted by the fat body in response to dietary protein, remains unknown. However, explant CNS culture experiments demonstrated that the FBDM can act directly on the CNS to bring about neuroblast reactivation (Britton, 1998). This study has identified the surface glia as a key relay in the nutritional control of neuroblast proliferation. If the receptor protein(s) that controls glial dILP expression/secretion can be identified, then, by extension, it might be possible to identify the FBDM and approach a comprehensive understanding of how neural stem cell proliferation is coupled to nutrition and organism-wide growth (Chell, 2010).

Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila

Developing animals survive periods of starvation by protecting the growth of critical organs at the expense of other tissues. This study used Drosophila to explore the as yet unknown mechanisms regulating this privileged tissue growth. As in mammals, it was observed in Drosophila that the CNS is more highly spared than other tissues during nutrient restriction (NR). Anaplastic lymphoma kinase (Alk) efficiently protects neural progenitor (neuroblast) growth against reductions in amino acids and insulin-like peptides during NR via two mechanisms. First, Alk suppresses the growth requirement for amino acid sensing via Slimfast/Rheb/TOR complex 1. And second, Alk, rather than insulin-like receptor, primarily activates PI3-kinase. Alk maintains PI3-kinase signaling during NR as its ligand, Jelly belly (Jeb), is constitutively expressed from a glial cell niche surrounding neuroblasts. Together, these findings identify a brain-sparing mechanism that shares some regulatory features with the starvation-resistant growth programs of mammalian tumors (Cheng, 2011).

This study found that CNS progenitors are able to continue growing at their normal rate under fasting conditions severe enough to shut down all net body growth. Jeb/Alk signaling was identified as a central regulator of this brain sparing, promoting tissue-specific modifications in TOR/PI3K signaling that protect growth against reduced amino acid and Ilp concentrations. These findings highlight that a 'one size fits all' wiring diagram of the TOR/PI3K network should not be extrapolated between different cell types without experimental evidence. The two molecular mechanisms by which Jeb/Alk signaling confers brain sparing is discussed, and how they may be integrated into an overall model for starvation-resistant CNS growth (Cheng, 2011).

One mechanism by which Alk spares the CNS is by suppressing the growth requirement for amino acid sensing via Slif, Rheb, and TORC1 components in neuroblast lineages. An important finding of this study is that in the presence of Alk signaling Tor has no detectable growth input (evidence from Tor clones), but in its absence (evidence from UAS-AlkDN; Tor clones) Tor reverts to its typical role as a positive regulator of both growth and proliferation. The growth requirement for Slif/TORC1 is clearly much less in the CNS than in other tissues such as the wing disc but a low-level input cannot be ruled out due to possible perdurance inherent in any clonal analysis. Although Slif, Rheb, Tor, and Raptor mutant neuroblast clones attain normal volume, this reflects increased cell numbers offset by reduced average cell size. Atypical suppression of proliferation by TORC1 has also been observed in wing discs, where partial inhibition with rapamycin advances G2/M progression (Cheng, 2011).

Alk signaling in neuroblast lineages does not override the growth requirements for all TOR pathway components. The downstream effectors S6k and 4E-BP retain functions as positive and negative growth regulators, respectively. 4E-BP appears to be particularly critical in the CNS as mutant animals have normal mass, but mutant neuroblast clones are twice their normal volume. In many tissues, 4E-BP is phosphorylated by nutrient-dependent TORC1 activity. In CNS progenitors, however, 4E-BP phosphorylation is regulated in an NR-resistant manner by Alk, not by TORC1. Hence, although the pathway linking Alk to 4E-BP is not yet clear, it makes an important contribution toward protecting CNS growth during fasting (Cheng, 2011).

A second mechanism by which Alk spares CNS growth is by maintaining PI3K signaling during NR. Alk suppresses or overrides the genetic requirement for InR in PI3K signaling, which may or may not involve the direct binding of intracellular domains as reported for human ALK and IGF-IR (Shi, 2009). Either way, in the CNS, glial Jeb expression stimulates Alk-dependent PI3K signaling and thus neural growth at similar levels during feeding and NR. In contrast, in tissues such as the salivary gland, where PI3K signaling is primarily dependent upon InR, falling insulin-like peptides concentrations during NR strongly reduce growth (Cheng, 2011).

The finding that Alk signals via PI3K during CNS growth differs from the Ras/MAPK transduction pathway described in Drosophila visceral muscle. However, a link between ALK and PI3K/Akt/Foxo signaling during growth is well documented in humans, both in glioblastomas and in non-Hodgkin lymphoma. Similarities with mammals are less obvious with regard to Alk ligands, as there is no clear Jeb ortholog and human ALK can be activated, directly or indirectly, by the secreted factors Pleiotrophin and Midkine (Cheng, 2011).

A comparison of these results with those of previous studies indicates that CNS super sparing only becomes fully active at late larval stages. Earlier in larval life, dietary amino acids are essential for neuroblasts to re-enter the cell cycle after a period of quiescence. This nutrient-dependent reactivation involves a relay whereby Slif-dependent amino acid sensing in the fat body stimulates Ilp production from a glial cell niche (Sousa-Nunes, 2011). In turn, glial-derived Ilps activate InR and PI3K/TOR signaling in neuroblasts thus stimulating cell cycle re-entry. Hence, the relative importance of Ilps versus Jeb from the glial cell niche may change in line with the developmental transition of neuroblast growth from high to low nutrient sensitivity (Cheng, 2011).

The results of this study suggest a working model for super sparing in the late-larval CNS. Central to the model is that Jeb/Alk signaling suppresses Slif/ RagA/Rheb/TORC1, InR, and 4E-BP functions and maintains S6k and PI3K activation, thus freeing CNS growth from the high dependence upon amino acid sensing and Ilps that exists in other organs. The CNS also contrasts with other spared diploid tissues such as the wing disc, in which PI3K-dependent growth requires a positive Tor input but is kept in check by negative feedback from TORC1 and S6K. Alk is both necessary (in the CNS) and sufficient (in the salivary gland) to promote organ growth during fasting. However, both Alk manipulations produce organ-sparing percentages intermediate between the 2% salivary gland and the 96% neuroblast values, arguing that other processes may also contribute. For example, some Drosophila tissues synthesize local sources of Ilps that could be more NR resistant than the systemic supply from the IPCs. In mammals, this type of mechanism may contribute to brain sparing as it has been observed that IGF-I messenger RNA (mRNA) levels in the postnatal CNS are highly buffered against NR. It will also be worthwhile exploring whether mammalian neural growth and brain sparing involve Alk and/or atypical TOR signaling. In this regard, it is intriguing that several studies show that activating mutations within the kinase domain of human ALK are associated with childhood neuroblastomas. In addition, fetal growth of the mouse brain was recently reported to be resistant to loss of function of TORC1. Finally, a comparison between the current findings and those of a cancer study, highlights that insulin/IGF independence and constitutive PI3K activity are features of NR-resistant growth in contexts as diverse as insect CNS development and human tumorigenesis (Cheng, 2011).

Insulin signals control the competence of the Drosophila female germline stem cell niche to respond to Notch ligands

Adult stem cells reside in specialized microenvironments, or niches, that are essential for their function in vivo. Stem cells are physically attached to the niche, which provides secreted factors that promote their self-renewal and proliferation. Despite intense research on the role of the niche in regulating stem cell function, much less is known about how the niche itself is controlled. Previous work has shown that insulin signals directly stimulate germline stem cell (GSC) division and indirectly promote GSC maintenance via the niche in Drosophila. Insulin-like peptides are required for maintenance of cap cells (a major component of the niche that are directly attached to GSCs through E-cadherin) via modulation of Notch signaling, and they also control attachment of GSCs to cap cells and E-cadherin levels at the cap cell-GSC junction. This study has further dissected the molecular and cellular mechanisms underlying these processes. Insulin and Notch ligands were shown to directly stimulate cap cells to maintain their numbers and indirectly promote GSC maintenance. It is also reported that insulin signaling, via phosphoinositide 3-kinase and FOXO, intrinsically controls the competence of cap cells to respond to Notch ligands and thereby be maintained. Contrary to a previous report, it was also found that Notch ligands originated in GSCs are not required either for Notch activation in the GSC niche, or for cap cell or GSC maintenance. Instead, the niche itself produces ligands that activate Notch signaling within cap cells, promoting stability of the GSC niche. Finally, insulin signals control cap cell-GSC attachment independently of their role in Notch signaling. These results are potentially relevant to many systems in which Notch signaling modulates stem cells and demonstrate that complex interactions between local and systemic signals are required for proper stem cell niche function (Hsu, 2011).

The Notch pathway plays a central role in many stem cell systems, and how systemic signals impact Notch signaling in stem cell niches is a question of wide relevance to stem cell biology. Notch controls cap cell number in the Drosophila female GSC niche, and recent studies showed that insulin-like peptides control Notch signaling in the niche (Hsu, 2009), although the underlying cellular mechanisms remained unclear. This study dissected the specific cellular requirements for Notch pathway components and the insulin receptor and reveals that insulin signaling controls cell–cell communication via Notch signaling within the niche (Hsu, 2011).

To summarize, from this study in combination with previous work, a fairly complex model emerges of how insulin-like peptides -- systemic signals influenced by diet -- impact the function of GSCs and their niche through multiple mechanisms. In adult females under favorable nutritional conditions, insulin-like peptides signal directly to GSCs via PI3K to inhibit FOXO and thereby increase their division rates by promoting progression through G2. In parallel to this direct effect on GSC proliferation, insulin-like peptides also act directly on cap cells (a major cellular component of the GSC niche) to control two separate processes. Stimulation of the insulin pathway, also via PI3K inhibition of FOXO, within cap cells intrinsically increase their responsiveness to the Notch ligand Delta (likely at a step upstream of nuclear translocation of the intracellular domain of Notch), which is likely produced by neighboring cap cells. (A similar process likely occurs during niche formation in larval/pupal stages, although in this case, Delta produced in basal terminal filament cells clearly contributes to the specification of cap cells.) Notch signaling within cap cells leads to their maintenance and, indirectly, to GSC maintenance. Independently of its effect on Notch signaling, insulin/PI3K/FOXO pathway activation in cap cells intrinsically promotes stronger cap cell-GSC adhesion (presumably via E-cadherin; Hsu, 2009), which also promotes GSC maintenance. Further, aging also appears to influence insulin signaling levels in Drosophila females (Hsu, 2009), suggesting that physiological changes caused by diverse factors can impinge on this GSC regulatory network. Together, these studies underscore the importance of investigating how whole organismal physiology impacts stem cell function via effects on stem cells and on their niche, potentially via changes in local signaling (Hsu, 2011).

Notch signaling requires direct cell-cell contact because Notch ligands are membrane-bound proteins that induce Notch activation in neighboring cells. In addition to transactivating Notch in adjacent cells, the Notch ligand Delta also inhibits Notch in cis, thus creating a potent switch between high Delta expression/low Notch activity and high Notch activity/low Delta expression (Sprinzak, 2010). Differential Notch activation often underlies binary cell fate decisions. For example, during Drosophila sensory organ development, cells with high levels of Delta and low Notch activity become neurons, while those with elevated Notch activity and low Delta become epidermal cells (Hsu, 2011).

In the Drosophila GSC niche, Notch activity is detected in all cap cells, and Dl-lacZ is expressed in all terminal filament cells. A subset of cap cells also expresses Dl-lacZ, suggesting that some cap cells may express Delta and have high Notch activity simultaneously. The basal terminal filament cell, in which Dl is required for cap cell formation, does not contact all cap cells directly, and it was also found that Dl and Ser are not required within GSCs for cap cell formation or maintenance. It is therefore proposed that cap cells may signal to each other via Delta to activate Notch signaling, and that, in cap cells, Delta might not consistently act in cis to inhibit Notch activation (Hsu, 2011).

The observation that a subset of cap cells can express Dl-lacZ and Notch activity simultaneously is consistent with recent findings. Human eosinophils express both Notch and its ligands, and autocrine Notch signaling controls their migration and survival (Radke, 2009). Similarly, Notch is co-expressed with its ligands in rat hepatocytes following partial hepatectomy and also in normal human breast cells, although it is unclear if autocrine signaling occurs. It is therefore conceivable that Delta expressed in cap cells may stimulate Notch signaling via both paracrine and autocrine manners (Hsu, 2011).

Alternatively, Notch ligands might be secreted from terminal filament cells to stimulate Notch signaling in all cap cells and thereby promote their maintenance. In fact, a soluble form of Delta capable of stimulating Notch has been identified in Drosophila S2 cell cultures, and the ADAM disintegrin metalloprotease Kusbanian is required for the production of soluble Delta in culture. Further, Dl and kuzbanian genetically interact, raising the possibility that soluble forms of ligands might modulate Notch signaling in vivo (Hsu, 2011).

neur encodes an E3 ubiquitin ligase that mediates the endocytosis of Notch ligands in signal-sending cells, thereby enhancing their signaling strength. Contrary to a previous report, this study found no evidence that Notch ligands produced from GSCs are required for self-renewal. In contrast, neur is intrinsically required for GSC maintenance. Similarly, in the Drosophila testis, neur, but not Dl and Ser, is required for GSC maintenance, further indicating that Neuralized maintains GSCs via a Notch-independent pathway (Hsu, 2011).

neur mutant cysts exhibit large and highly branched fusomes, another Notch-independent phenotype. In principle, this aberrant fusome morphology might result from a defect in fusome growth and/or partitioning, or be secondary to an excessive number of cyst division rounds. Nevertheless, the close association of some of these abnormal fusomes with the cap cell interface suggests that fusome defects might lead to GSC loss. Ubiquitination regulates many processes, including protein degradation and vesicular trafficking. It is therefore possible that Neuralized ubiquitinates specific substrates that regulate fusome-related vesicular trafficking during cyst division. Future studies should test whether E3 ligase activity is indeed required for the role of neur in early germline cysts, identify key ubiquitination targets, and elucidate the molecular mechanisms they regulate (Hsu, 2011).

Under low insulin signaling, the FOXO transcriptional factor is required for extended longevity, reduced rates of proliferation, and stress resistance, among other processes. FOXOs are conserved from yeast to humans, and they control many target genes, different subsets of which modulate distinct processes. Drosophila FOXO negatively controls GSC division when insulin signaling is low (Hsu, 2008). It was also shown that insulin signaling modulates niche-stem cell interactions and Notch signaling in the niche (to control cap cell number), and that insulin signaling declines as females become older, leading to stem cell loss (Hsu, 2009). This study has shown that FOXO is required to negatively regulate Notch signaling within cap cells under low insulin activity and that FOXO also modulates the physical interaction between cap cells and GSCs. The multiplicity of FOXO roles in stem cell regulation is further underscored by studies in other stem cell systems. For example, FOXOs regulate several processes, including cell cycle progression, oxidative stress, and apoptosis, in the hematopoietic stem cell compartment, thereby influencing stem cell number and activity. It will be important to investigate how the specificity of FOXO is controlled and also whether or not FOXO regulates other stem cell niches, perhaps acting as a mediator of changes in niche size and/or activity during aging or cancer development (Hsu, 2011).

This study suggests a potentially novel mechanism by which the Notch and insulin pathways interact. In the Drosophila female GSC niche, insulin signaling does not control ligand transcription, and it is not required for ligand function (i.e., Dl is required in basal terminal filament cells during cap cell formation, but InR is not). Instead, both InR and N are cell autonomously required for cap cell maintenance, and insulin receptor function (via repression of FOXO) is required for proper Notch signaling. Expression of the intracellular domain of Notch rescues the low cap cell and GSC numbers of InR mutants (Hsu, 2009), and ovarian Notch expression does not appear altered in InR mutants. Therefore, it is speculated that FOXO inhibits the ability of cap cells to respond to Notch ligands by regulating a target that negatively regulates the series of proteolytic events responsible for the release of the intracellular domain of Notch. It cannot, however, be rulef out that Notch and FOXO normally interact at the level of target gene regulation but that overexpression of the intracellular domain of Notch overrides the normal inhibition by FOXO (Hsu, 2011).

These findings contrast with other types of interactions between FOXO and Notch that have been reported. During muscle differentiation in myoblast cultures, FOXO promotes (instead of antagonizing) Notch activity via a physical interaction that leads to activation of Notch target genes. Positive interactions between Notch and PI3K signaling have also been reported. Specifically, activation of the PI3K pathway potentiates Notch-dependent responses in CHO cells, T-cells, and hippocampal neurons. The suggested mechanism, however, involves the inactivation of GSK3 by Akt phosphorylation upstream of FOXO, which is distinct from the involvement of FOXO in the insulin-Notch signaling interaction within the GSC niche. These examples illustrate the diversity of modes of interaction between Notch and insulin signaling. It is conceivable that the positive interaction that is describe between insulin and Notch signaling pathways in the GSC niche may occur in other stem cell niches (Hsu, 2011).

Deregulated Notch signaling is associated with many types of cancers and, in some cases, it is thought that altered Notch signaling promotes cancer development by overstimulating the self-renewal of normal stem cells (Wang, 2009). Hyperactivation of insulin/IGF pathway is also linked to increased cancer risk and poor cancer prognosis. The Notch and insulin/IGF pathways have been reported to interact in cancerous cells via yet another mechanism. Specifically, upregulation of the Notch ligand Jagged 1 leads to PI3K activation in human papillomavirus-induced cancer lines. It is speculated that additional types of interactions between Notch and insulin/IGF signaling, such as the positive regulation of Notch activity by the insulin/PI3K/FOXO pathway that occurs in the Drosophila GSC niche, may also contribute to cancer progression (Hsu, 2011).

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

Genetic studies in Drosophila reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches this study tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-β (GSK3β) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. This study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level it was found that both these pathways converge at GSK3β to control Myc protein stability, while genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia (Parisi, 2011).

Previous studies in vertebrates have indicated a critical function for Myc downstream of growth factor signaling including insulin-like growth factor, insulin and TOR pathways. In Drosophila, despite a few notes that Myc transcriptional activity acts downstream of insulin and TOR pathways, no clear molecular mechanisms linking these pathways to Myc have been elucidated yet (Parisi, 2011).

It has been demonstrated that inhibition of GSK3β prevents Myc degradation by the proteasome pathway. This study further unravels the pathways that control Myc protein stability and shows that signaling by insulin and TOR induce Myc protein accumulation by regulating GSK3β activity in S2 cells. GSK3β is a constitutively active kinase that is regulated by multiple signals and controls numerous cellular processes. With the biochemical data it is proposed that GSK3β acts as a common point where insulin and TOR signaling converge to regulate Myc protein stability (see Model showing the proposed relationship between Myc and the insulin and TOR signaling pathways). In particular, activation of insulin signaling was shown to induce activation of Akt, an event that is accompanied by GSK3β phosphorylation on Ser 9 that causes its inactivation and Myc protein to stabilize. Interestingly, insulin-induced Myc protein accumulation, when GSK3β activity was blocked by the presence of LiCl or by expression of GSK3β-KD, was similar to that obtained with insulin alone. Since it was shown that activation of insulin signaling leads to GSK3β inhibition and to an increase in Myc protein, if insulin and GSK3β signaling were acting independently, it would be expected that activation of insulin signaling concomitantly with the inhibition of GSK3β activity would result in a higher level of Myc than that obtained with insulin or LiCl alone. The results instead showed a similar level of Myc protein accumulation with insulin in the presence of GSK3β inhibitors as compared to insulin alone, supporting the hypothesis that GSK3β and insulin signaling, at least in the current experimental condition, depend on each other in the mechanism that regulates Myc protein stability (Parisi, 2011).

In a similar biochemical approach, the effect of AAs was analyzed on Myc protein stability and how TOR signaling is linked to mechanisms that inactivate GSK3β to stabilize Myc protein in S2 cells. In these experiments it was possible to demonstrate that treatment with amino acids (AAs) increased Myc protein stability, and it was also shown that treatment with rapamycin, an inhibitor of TORC1, reduced insulin-induced Myc upregulation. The reduction of Myc protein accumulation by rapamycin was blocked by inhibition of the proteasome pathway, linking TOR signaling to the pathway that controls Myc protein stability. TORC1 is a central node for the regulation of anabolic and catabolic processes and contains the central enzyme Rheb-GTPase, which responds to amino acids by activating TOR kinase to induce phosphorylation of p70-S6K and 4E-BP1. Analysis of the molecular mechanisms that act downstream of TOR to regulate Myc stability shows that AA treatment induces p70-S6K to phosphorylate GSK3β on Ser 9, an event that results in its inactivation and accumulation of Myc protein (Parisi, 2011).

Reducing GSK3β activity with LiCl, in medium lacking AAs, resulted in a slight increase in Myc protein levels. Adding back AAs lead to a substantial increase in Myc protein levels, which did not further increase when AAs where added to cells in the presence of the GSK3β inhibitor LiCl. These events were accompanied by phosphorylation of S6K on Thr 398, which correlated with phosphorylation of GSK3β on Ser 9. From these experiments it is concluded that TOR signaling also converges to inhibit GSK3β activity to regulate Myc protein stability. However, it has to be pointed out that since AAs alone increased Myc protein levels to a higher extent than that observed with LiCl alone, the experiments also suggest that Myc protein stability by TOR signaling is not solely directed through the inhibition of GSK3β activity, but other events and/or pathways contribute to Myc regulation. In conclusion, the biochemical experiments demonstrate that GSK3β acts downstream of insulin and TOR pathways to control Myc stability, however it cannot be excluded that other pathways may control Myc protein stability upon insulin and amino acids stimulation in S2 cells (Parisi, 2011).

Reduction of insulin and TOR signaling in vivo reduces cell size and proliferation, and clones mutant for chico, the Drosophila orthologue of IRS1-4, or for components of TOR signaling, are smaller due a reduction in size and the number of cells. The experiments showed that reducing insulin signaling by expression of PTEN or using TORTED, a dominant negative form of TOR, decreased Myc protein levels in clones of epithelial cells of the wing imaginal discs, while the opposite was true when these signals were activated using Dp110 or RhebAV4 . Those experiments suggested that the mechanism of regulation of Myc protein by insulin and TOR pathways was conserved also in vivo in epithelial cells of the larval imaginal discs (Parisi, 2011).

During these experiments it was also noted that Myc protein was induced in the cells surrounding and bordering the clones (non-autonomously), particularly when clones where positioned along the dorsal-ventral axis of the wing disc. This upregulation of Myc protein was not restricted to components of the insulin signaling pathway since it was also observed in cells surrounding the clones mutant for components of the Hippo pathway or for the tumor suppressor lethal giant larvae (lgl), which upregulates Myc protein cell-autonomously. It is suspected that this non-autonomous regulation of Myc may be induced by a novel mechanism that controls proliferation of cells when 'growth' is unbalanced. It can be speculated that clones with different growth rates, caused by different Myc levels, might secrete factors to induce Myc expression in neighboring cells. As a consequence, these Myc-expressing cells will speed up their growth rate in an attempt to maintain proliferation and tissue homeostasis. Further analysis is required to identify the mechanisms responsible for this effect (Parisi, 2011).

In order to distinguish if Myc activity was required downstream of insulin and TOR signaling to induce growth, a genetic analysis was performed. The ability to induce growth and proliferation was measured in the eye by measuring the size and number of the ommatidia from animals expressing members of the insulin and TOR pathways in different dm genetic background (dm+, dmP0 and dm4). The data showed that Dp110 increased the size and number of the ommatidia, however only the alteration in the total number was dependent on dm levels. These data suggest that Myc is required downstream of insulin pathway to achieve the proper number of ommatidia. However, when insulin signaling was reduced by PTEN, a significant decrease in the size of ommatidia was seen and it was dependent on dm expression levels, suggesting that Myc activity is limiting for ommatidial size and number. Activation of TOR signaling induces growth, and the genetic analysis showed that Myc significantly contributes to the size of the ommatidial cells thus suggesting that Myc acts downstream of TOR pathway to control growth (Parisi, 2011).

Recent genomic analysis showed a strong correlation between the targets of Myc and those of the TOR pathway, implying that they may share common targets. In support of this observation, mosaic analysis with a repressible cell marker (MARCM) experiments in the developing wing disc showed that overexpression of Myc partially rescues the growth disadvantage of clones mutant for the hypomorphic Rheb7A1 allele, further supporting the idea that Myc acts downstream of TOR to activate targets that control growth in these clones (Parisi, 2011).

The genetic interaction revealed a stronger dependence on Myc expression when Rheb was used as opposed to S6K. A possible explanation for this difference could lie in the fact that S6K is not capable of auto-activation of its kinase domain unless stimulated by TOR kinase. TOR activity is dependent on its upstream activator Rheb; consequently the enzymatic activity of the Rheb/GTPase is the limiting factor that influences S6K phosphorylation and therefore capable of maximizing its activity (Parisi, 2011).

Interestingly, these experiments also showed that activation of TOR signaling has a negative effect on the number of ommatidia, and this correlates with the ability of RhebAV4 to induce cell death during the development of the eye imaginal disc. Rheb-induced cell death was rescued in a dmP0 mutant background, which leading to the speculation that 'excessive' protein synthesis, triggered by overexpression of TOR signaling, could elicit a Myc-dependent stress response, which induces apoptosis. Alternatively, high protein synthesis could result in an enrichment of misfolded proteins that may result in a stress response and induces cell death. Further analysis is required to delineate the mechanisms underlying this process (Parisi, 2011).

This analyses provide novel genetic and biochemical evidences supporting a role for Myc in the integration of the insulin and TOR pathway during the control of growth, and highlights the role of GSK3β in this signaling. It was found that insulin signaling inactivates GSK3β to control Myc protein stability, and a similar biochemical regulation is also shared by activation of the TOR pathways. In support of this data, a recent genomic analysis in whole larvae showed a strong correlation between the targets of Myc and those of the TOR pathway; however, less overlap was found between the targets of Myc and those of PI3K signaling (Parisi, 2011).

Statistical analysis applied to the genetic interaction experiments revealed that, in the Drosophila eye, proliferation induced by activation of the insulin pathway is sensitive to variations in Myc levels, while a significant interaction was seen mostly when TOR increased cell size. The data therefore suggests that there is a correlation between Myc and the InR signaling and it is expected that the InR pathway also shares some transcriptional targets with Myc. Indeed, an overlap was found between the targets induced by insulin and Myc in Drosophila S2 cells and these targets have also been reported in transcriptome analyses in the fat body upon nutritional stress, suggesting that Myc acts downstream of InR/PI3K and TOR signaling and that this interaction might be specific to some tissues or in a particular metabolic state of the cell (Parisi, 2011).

ole of the insulin/Tor signaling network in starvation-induced programmed cell death in Drosophila oogenesis

Amino-acid starvation leads to an inhibition of cellular proliferation and the induction of programmed cell death (PCD) in the Drosophila ovary. Disruption of insulin signaling has been shown to inhibit the progression of oogenesis, but it is unclear whether this phenotype mimics starvation. This study investigated whether the insulin-mediated phosphoinositide kinase-3 pathway regulates PCD in mid oogenesis. It was reasoned that under well-fed conditions, disruption of positive components of the insulin signaling pathway within the germline would mimic starvation and produce degenerating egg chambers. Surprisingly, mutants did not mimic starvation but instead produced many abnormal egg chambers in which the somatic follicle cells disappeared and the germline persisted. These abnormal egg chambers did not show an induction of caspases and lysosomes like that observed in wild-type (WT) degenerating egg chambers. Egg chambers from insulin signaling mutants were resistant to starvation-induced PCD, indicating that a complete block in insulin-signaling prevents the proper response to starvation. However, target of rapamycin (Tor) mutants did show a phenotype that mimicked WT starvation-induced PCD, indicating an insulin independent regulation of PCD via Tor signaling. These results suggest that inhibition of the insulin signaling pathway is not sufficient to regulate starvation-induced PCD in mid oogenesis. Furthermore, starvation-induced PCD is regulated by Tor signaling converging with the canonical insulin signaling pathway (Pritchett, 2012).

These results indicate that the insulin signaling pathway is an important factor for cell survival in the Drosophila ovary, similar to its role in mammals. Mammalian ovaries cultured in serum-free media show an induction in apoptotic and autophagic PCD. Survival of mouse primordial follicles requires PI3K, phosphoinositide-dependent protein kinase-1 (PDK1), and S6k1, which are members of the insulin signaling cascade. Treatment with rapamycin, a drug known to block Tor activity, inhibits oocyte growth in cultured Drosophila and mammalian ovaries, and leads to apoptosis and autophagy. Taken together, these findings suggest an evolutionarily conserved role for insulin and Tor signaling in promoting survival in the ovary. Furthermore, characterization of starvation-induced PCD in the Drosophila ovary may give insight into the mechanisms of degeneration of defective oocytes in mammalian systems during reproductive aging and fertility disorders (Pritchett, 2012).

Tight coordination of growth and differentiation between germline and soma provides robustness for Drosophila egg development

Organs often need to coordinate the growth of distinct tissues during their development. This study analyzed the coordination between germline cysts and the surrounding follicular epithelium during Drosophila oogenesis. Genetic manipulations of the growth rate of both germline and somatic cells influence the growth of the other tissue accordingly. Growth coordination is therefore ensured by a precise, two-way, intrinsic communication. This coordination tends to maintain constant epithelial cell shape, ensuring tissue homeostasis. Moreover, this intrinsic growth coordination mechanism also provides cell differentiation synchronization. Among growth regulators, PI3-kinase and TORC1 also influence differentiation timing cell-autonomously. However, these two pathways are not regulated by the growth of the adjacent tissue, indicating that their function reflects an extrinsic and systemic influence. Altogether, these results reveal an integrated and particularly robust mechanism ensuring the spatial and temporal coordination of tissue size, cell size, and cell differentiation for the proper development of two adjacent tissues (Vachias, 2014: PubMed).

Several main conclusions can be drawn from this work. First, in each follicle, growth is intrinsically coordinated between the two tissues. Second, this growth control tends to optimize cell shape in the epithelium. This is likely to be representative of the development of many epithelia where cell shape must be maintained because it is essential for the function of the tissue. In the third place, growth control has a very important impact on differentiation timing in each tissue. Furthermore, several growth pathways can cell-autonomously influence differentiation rate but are not regulated by the adjacent tissue, indicating that they only respond to extrinsic cues. Finally, as a whole, this study reveals the robustness of the spatiotemporal pattern allowing the production of mature eggs with a normal shape and a normal size. At least two examples based on Pten somatic clones can illustrate this robustness. WT border cells migrate perfectly 'on time' in a follicle in which mutant somatic cells have induced a faster germline development. Second, a WT looking mature egg can be found in the middle of an ovariole, suggesting that all developmental steps have been faster but correctly orchestrated. This robustness probably reflects the fact that final egg size is constant, that most of the developmental steps have to occur at a specific size, and that differentiation is able to block growth when the definitive egg size is reached. These observations raise the question as to how the differentiation program regulates growth and especially growth arrest in each follicle (Vachias, 2014).

These results indicate a two-way communication between the germline and the soma to ensure their coordination. It was also observed that somatic cells can influence other somatic cells but, importantly, that such an effect depends on the relay of the germ cells. This result suggests that coordination is achieved by different signals depending on the tissue. The soma and germline could communicate via the secretion of growth factors controlling the adjacent tissue, though obvious candidates were excluded. An alternative explanation would be that, as it is proposed in mammals, the two tissues are interdependent for specific metabolites, although it would be independent of TORC1, a classical sensor of metabolic activity. Finally, an attractive hypothesis would be that growth regulation between the soma and the germline depends on a mechanical steady state. Germline growth creates a tension on the follicle cell, leading to the proposal that this tension could trigger epithelial growth. If so, it would also mean that follicle cells provide a mechanical strain limiting germline growth. The mechanical control of growth in epithelial cells is usually devoted to the Hippo pathway, which is not involved in this instance. Thus, this work does not allow favoring one or the other of these nonexclusive mechanisms (Vachias, 2014).

Altogether, these results highlight several dimensions of coordination between cell growth, cell shape, and cell identity and all this between two distinct tissues. These different functional links offer a highly robust program in space and time. The relevance for such robustness has been very recently highlighted because it probably confers the reproducibility on embryonic development. Since usual pathways controlling growth are not involved in this two-way communication, this multidimensional coordination will be a useful framework for identifying molecular actors ensuring tissue homeostasis in the recurrent context of the development of two adjacent tissues (Vachias, 2014).

Presynaptic PI3K activity triggers the formation of glutamate receptors at neuromuscular terminals of Drosophila

Synapse transmission depends on the precise structural and functional assembly between pre- and postsynaptic elements. This tightly regulated interaction has been thoroughly characterised in vivo in the Drosophila glutamatergic larval neuromuscular junction (NMJ) synapse, a suitable model to explore synapse formation, dynamics and plasticity. Previous findings have demonstrated that presynaptic upregulation of phosphoinositide 3-kinase (PI3K) increases synapse number, generating new functional contacts and eliciting changes in behaviour. This study shows that genetically driven overexpression of PI3K in the presynaptic element also leads to a correlated increase in the levels of glutamate receptor (GluRII) subunits and the number of postsynaptic densities (PSDs), without altering GluRII formation and assembly dynamics. In addition to GluRIIs, presynaptic PI3K activity also modifies the expression of the postsynaptic protein Discs large (Dlg). Remarkably, PI3K specifically overexpressed in the final larval stages is sufficient for the formation of NMJ synapses. No differences in the number of synapses and PSDs were detected when PI3K was selectively expressed in the postsynaptic compartment. Taken together, these results demonstrate that PI3K-dependent synaptogenesis plays an instructive role in PSD formation and growth from the presynaptic side (Jordan-Alvarez, 2012).

Persistent high levels of PI3K activity are necessary not only for synapse formation, but also for its subsequent maintenance and functionality (Cuesto, 2011; Martin-Pena, 2006). Unfortunately, however, the lack of a valuable PI3K antibody in Drosophila precludes an unequivocal detection of the protein in the pre- or postsynaptic compartments. Electrophysiological recordings from larval motor neurons overexpressing PI3K indicated an increase in evoked EPSP size and increments in both MEPP frequency and MEPP amplitude (Martin-Pena, 2006). These features could be accounted for by either a presynaptic or a postsynaptic role of PI3K. Additionally, rat hippocampal cultured neurons stimulated with PTD4-PI3KAc, a PI3K-activating transduction peptide, showed an increase in basal mEPSC frequency without any augmentation in mEPSC amplitude (Cuesto, 2011), consistent with an increase in functional synapses without changes in the postsynaptic receptor properties. Moreover, a postsynaptic role of PI3K has been demonstrated on the maintenance of the physiological PIP3 levels at postsynaptic densities, most likely by controlling AMPA receptor turnover (Arendt, 2010). Thus, to date, both immunochemical and electrophysiological findings failed to fully assign the synaptic compartment from which PI3K is causing these phenotypes (Jordan-Alvarez, 2012).

The data reported in this study demonstrate that postsynaptic GluRIIs respond to PI3K changes induced in the presynaptic side of the developing NMJ. It has been previously reported that immature PSDs typically are dominated by GluRIIA, being subsequently balanced by GluRIIB incorporation during PSD and synapse maturation (Schmid, 2008). This balance modifies the electrical properties of the glutamate receptor and seems to be directly correlated to increases of the presynaptic levels of Brp, crucial for mature presynaptic glutamate release (Schmid, 2008) and also for structural integrity of the active zone. Thus, the integration of both pre- and postsynaptic elements controls efficiently the number of synapses and, hence, PSDs per NMJs. This study has found that presynaptic, but not postsynaptic, PI3K overexpression yields to an augmentation of all NMJ synaptic markers examined. By contrast, presynaptic PI3K downregulation does alter neither GluRIIA) nor the two Dlg isoforms tested. However, the reduction of PI3K activity using PI3KDN gives rise to a decrease in both synapse and PSD (quantified by GluRIID immunostaining) numbers. This apparent discrepancy is probably due to the fact that the mechanism of attenuating PI3K activity with a dominant negative (PI3KDN) is not fully efficient to generate a detectable reduction in all GluRII subunits or Dlg protein levels (Jordan-Alvarez, 2012).

Notably, this study also found changes in the number of PSDs along larval development and NMJ maturation when PI3K overexpression was selectively restricted to third instar larvae. The data also indicate a timing of around 24 hours to generate increases in PSD number. However, no differences were found in synapse number at 24 hours, although there was a tendency towards increment in PI3K versus control larvae. It is reasonable to assume that PI3K needs time to be transcribed, translated and accumulated in the NMJ to generate new synapses. Thus, the temperature shift, which allows PI3K expression only in the last 18 hours, is insufficient for the generation of fully mature synapses, detected by nc82 antibody, but enough to detect immature PSDs by GluRIIA immunostaining. Previous data have shown a synaptic half-life of around 24 h in larval NMJ, but also in fully differentiated brain neurons. In mammals, dendritic spines are functional within a day after induction of long-term plasticity (LTP) in hippocampal slices (Jordan-Alvarez, 2012).

The dynamics of GluRIIA incorporation into PSDs is not affected by PI3K. This feature could be explained by a PI3K role triggering the formation of additional new nascent synapses by increasing the number of sites where active zones will be formed, leaving unaffected the time of GluR incorporation at individual PSD. In turn, the number of PSDs could be incremented due to the recruitment of glutamate receptors from pools dispersed over the whole muscle cell membrane that could be able to respond to the PI3K-dependent increase of suitable synaptic sites. Previous findings obtained in excitatory synapses in vertebrates indicate that the PIP3 pathway is linked to AMPAR insertion in the membrane. In hippocampal neurons, PI3K localises and directly binds to the cytosolic C-terminus of the AMPAR (Man, 2003) and this PI3K-AMPAR association plays a significant role in sustaining synaptic transmission (Arendt, 2010; Jordan-Alvarez, 2012 and references therein).

Different mechanisms could account for the PI3K effects on postsynaptic proteins revealed in this study: first, a higher expression of glutamate receptor subunits should be achieved by increased synthesis or by reduced local receptor protein degradation. Indeed, the regulation of both protein synthesis and turnover are key factors for synaptic terminal development and function at the Drosophila NMJ. Second, receptor subunits enter to the newly forming clusters from a diffuse pool of receptors, not by splitting from previously formed ones. This indicates a crucial role for protein trafficking, diffusion and clustering mechanisms in the developing NMJ. In this context, SYD-2/Liprin-alpha has been implicated in both pre- and postsynaptic assembly by interacting with a multitude of synaptic proteins and by regulating synaptic cargo transport guiding transport of active zone components. Liprin family proteins steer transport in axons and dendrites (e.g. of AMPA receptors) to support synaptic specialisations and play a key role in AZ assembly function. Moreover, the presynaptic AZ-localised RhoGAP DSyd-1 acts in a trans-synaptic manner, by targeting DLiprin-α to maturing AZs, and also defining the amount and composition of glutamate receptors (GluRs) accumulating at PSDs (Jordan-Alvarez, 2012).

The case of larval motor neurons described in this study has a precedent on the optic ganglia in which presynaptic photoreceptors determine the number of synapses established with the lamina neuron. In this study, the new data provide a molecular mechanism and highlight the instructive role of PI3K in the regulation of synapse number and postsynaptic proteins in the NMJ. Also, these data have paved the way to understand the trans-synaptic signals needed for the formation, maturation and dynamic regulation of the synapse (Jordan-Alvarez, 2012).

Effects of Mutation or Deletion

The human tumor suppressor gene PTEN gets its name from its biochemical function, its domain structure and its chromosomal location: PTEN stands for the combination of phosphatase and tensin homolog on chromosome 10. The lipid phosphatase function of PTEN places it in the middle of the insulin pathway, known to involve lipid signaling (Goberdhan, 1999; Huang, 1999). Drosophila Pten modulates cell size and consequently tissue mass by acting antagonistically to the lipid modifiying enzyme Phosphotidylinositol 3 kinase 92E, also known as Dp110, and its upstream activator Chico, an insulin receptor binding and signal transduction protein. All signals from the insulin receptor can be antagonized by Pten. In terms of its protein phosphatase function, mammalian PTEN targets focal adhesion kinase, a major effector of cytoskeletal function. Overexpression of wild-type mammalian PTEN and mutant PTEN that lacks lipid phosphatase activity can reduce levels of focal adhesion kinase (FAK: see Drosophila Focal adhesion kinase-like) phosphorylation and the formation of focal adhesions, thereby inhibiting cell migration and invasiveness.

Overexpression of Pten also produces enlargement of wing cells. Wild-type Pten cDNA is overexpressed in particular areas of the wing using the GAL4-UAS system. Initially flies carrying a dpp-GAL4 construct were used. This drives gene expression in cells that will normally populate the region between the third and fourth longitudinal wing veins (LIII and LIV). Overexpression of Pten reduces the size of these regions by nearly 25% compared with wild type. This is not a consequence of a general reduction in wing size in overexpressing flies, since an adjacent area of the wing between LIV and LV is essentially unaffected. The effect on wing area is similar to that produced by overexpression of Dp110D954A, a dominant-negative, kinase-dead version of Pi3K92E. The reduction is caused by both a decrease in cell size and cell number and is opposite of the effect of overexpressing an activated, membrane-associated form of Pi3K92E, Dp110-CAAX, in the same region. To test whether Pten's growth regulatory functions are primarily mediated by its effects on the insulin receptor-Pi3K92E signaling pathway and not by an independent signaling cascade, the genetic effects of Pten alleles were sought using mutant phenotypes associated with chico and Pi3K92E (Goberdhan, 1999).

Interestingly, both overexpression of a dominant negative form of Phosphotidylinositol 3 kinase 92E (also known as Dp110 or Pi3K92E) and mutations either in the Drosophila insulin receptor or in chico/IRS1-4 also reduce cell size as well as proliferation. Furthermore, overexpression of wild-type and activated forms of Pi3K92E produces similar size and proliferation defects as those seen in Pten mutant cells. These observations are consistent with a model in which growth is regulated in Drosophila by specific phosphoinositides whose levels are controlled by the balance of Pten and Pi3K92E activities. Pten and Pi3K92E. At 25°C, flies do not survive to adult when wild-type Pi3K92E is ectopically expressed by means eyeless-GAL4. Interestingly, this lethality can be rescued by coexpression of Pten. Furthermore, the small eye phenotype of Pten overexpression is suppressed by overexpression of wild-type Pi3K92E and enhanced by overexpression of dominant negative Pi3K92E. These results clearly indicate that Pten and Pi3K92E function antagonistically in Drosophila. The recent characterization of chico, a Drosophila IRS1-4 homolog, has shown that chico, Pi3K92E and Insulin receptor(Inr) act as positive elements in a Drosophila insulin signaling pathway to regulate cell proliferation and cell size (Huang, 1999).

Class IA phosphoinositide 3-kinases (PI 3-kinases) have been implicated in the regulation of several cellular processes including cell division, cell survival and protein synthesis. The size of Drosophila imaginal discs (epithelial structures that give rise to adult organs) is maintained by factors that can compensate for experimentally induced changes in these PI 3-kinase-regulated processes. However, overexpression of the gene encoding the Drosophila class IA PI 3-kinase, Dp110, in imaginal discs, results in enlarged adult organs. These observations have led to an investigation of the role of Dp100 and its adaptor, p60, in the control of imaginal disc cell size, cell number and organ size. Null mutations in Dp110 and p60 were generated and used to demonstrate that these genes are essential genes that are autonomously required for imaginal disc cells to achieve their normal adult size. In addition, modulating Dp110 activity increases or reduces cell size in the developing imaginal disc, and does so throughout the cell cycle. The inhibition of Dp110 activity reduces the rate of increase in cell number in the imaginal discs, suggesting that Dp110 normally promotes cell division and/or cell survival. Unlike direct manipulation of cell-cycle progression, manipulation of Dp110 activity in one compartment of the disc influences the size of that compartment and the size of the disc as a whole. It is concluded that during imaginal disc development, Dp110 and p60 regulate cell size, cell number and organ size. These results indicate that Dp110 and p60 signaling can affect growth in multiple ways: these observations have important implications for the function of signaling through class IA PI 3-kinases (Weinkove, 1999).

To investigate whether Dp110 and p60 are required within the imaginal discs themselves, mitotic clones of mutant cells were generated in heterozygous Dp110 or p60 flies by using the Flipase (Flp) and Flp recognition target (FRT) site-specific recombination system. Mutant phenotypes were examined in the adult eye, a highly ordered structure made up of repeated units or ommatidia, each of which contains the same pattern of differentiated photoreceptors and accessory cells. Both Dp110- and p60- eye clones form indented patches containing ommatidia that are reduced in size. The internal eye structure was examined using tangential sections in which Dp110- clones (-/-) were distinguished from their corresponding sister clones or twin-spots (+/+) and the surrounding heterozygous cells (+/-) by an absence of red pigment granules (Weinkove, 1999).

Dp110- cells, and to a lesser extent p60- cells, are significantly smaller in both cross-section and longitudinal-section than cells in the surrounding heterozygous and wild-type tissue. Thus, these experiments show that both Dp110 and p60 are autonomously required for eye cells to achieve their normal adult size. In addition, the mutant clones are consistently smaller than their twin-spots, suggesting that cell number as well as cell size might be reduced. Although reduced in size, the mutant photoreceptors can still differentiate and form rhabdomeres (which stain dark blue) by expanding and re-organizing their internal membranes. Furthermore, almost every ommatidium contains the wild-type number of photoreceptors, suggesting that Dp110 and p60 are not essential for photoreceptor differentiation. The characteristic trapezoid arrangement of photoreceptor rhabdomeres within each ommatidium and the orientation of the ommatidia relative to one another are generally wild type in p60- clones, but are sometimes disrupted in Dp110- clones. Together, these data clearly demonstrate that in Drosophila the class IA PI 3-kinase, Dp110, and its adaptor, p60, are required in a cell-autonomous manner for the same process: the attainment of normal cell size. The similarity of the mutant phenotypes for the two genes is consistent with the established role of the adaptor in the activation of class IA PI 3-kinases and represents the first direct comparison of the loss of function phenotypes of a class IA PI 3-kinase and its adaptor (Weinkove, 1999).

Thus Dp110 and p60 are necessary for adult eye cells to achieve their normal final size. A possible explanation for this requirement is that Dp110 drives growth solely in the final stages of eye development when the retinal cells increase in size after proliferation has ceased. Alternatively, Dp110 activity might be required for both proliferating and post-mitotic imaginal disc cells to achieve their normal size. To distinguish between these two possibilities, an investigation was carried out of the effect of modulating Dp110 activity on the size of the proliferating cells of third instar wing imaginal discs. Thus, Dp110 and p60 transgenes under the control of GAL4 upstream activating sequences (UASs) were expressed in clones of cells induced at random locations in wing imaginal discs. signaling through Dp110 was increased by overexpression of wild-type Dp110 and reduced by overexpression of a catalytically inactive and hence dominant-negative version of Dp110 (Dp110D954A). Greater inhibition of Dp110 signaling was achieved by overexpression of wild-type p60 or of p60 with part of the Dp110-binding site deleted. Like the mammalian class IA PI 3-kinase adaptors, p60 and deltap60 inhibit class IA PI 3-kinase signaling when overexpressed, presumably by competing with endogenous Dp110 and p60 complexes for binding sites on upstream activators. Dp110 expression dramatically increases imaginal disc cell size. In contrast, the expression of Dp110D954A slightly reduces cell size whereas the expression of p60 or deltap60 results in a more dramatic reduction in cell size. Thus, modulating Dp110 activity alters the size of proliferating cells in the third instar disc epithelium. To investigate this effect more closely, the sizes of Dp110-expressing and deltap60-expressing cells were examined at different stages of the cell cycle. This analysis demonstrated that Dp110-expressing cells are larger than control cells during both the G1 phase of the cell cycle and the S + G2. Conversely, deltap60-expressing cells are reduced in size both in G1 and S + G2. Thus, Dp110 regulates cell size throughout the imaginal disc cell cycle (Weinkove, 1999).

The increased size of Dp110-expressing cells might result either from an inhibition of cell division, or from accelerated biosynthesis. To distinguish between these two possibilities, the effect of Dp110 expression on cell number was examined. This analysis reveals that Dp110 expression does not alter the rate of increase in cell number. Thus, Dp110-expressing cells are larger because of increased growth and not because of the inhibition of cell division. Furthermore, this result demonstrates that, as far as can be detected with this technique, overexpression of Dp110 is not sufficient to increase cell number. In contrast, deltap60, p60, and to a lesser extent Dp110D954A expression, each reduce the rate of increase in cell number. Thus, attenuating Dp110 activity reduces cell size by reducing growth as opposed to increasing cell division. Taken together, these data demonstrate that the activity of Dp110 is necessary for cells to achieve their normal size and number (Weinkove, 1999).

During flow cytometry analysis, it was noted that, compared with the control cell populations, a greater proportion of the Dp110-expressing cells are in the G2 phase of the cell cycle whereas a greater proportion of the deltap60-expressing cells are in the G1 phase. These observations are consistent with an increase in Dp110 activity increasing the rate of progression of cells through the G1/S transition but not through the G2/M transition of the cell cycle (Weinkove, 1999).

Previous experiments, which utilized GAL4 under the control of the engrailed promotor (En-GAL4) to drive the expression of cell-cycle regulators in the posterior compartment of the wing imaginal disc, have shown that activation or inhibition of cell division in one compartment increases or reduces cell number without affecting the size of that compartment. Rather, that compartment contains more smaller cells or fewer bigger cells and growth is unaffected. These results suggest that imaginal disc growth is not regulated through the cell cycle but by an additional mechanism that is dominant over the cell cycle. Over-expression of Dp110 increases adult wing and eye size, whereas overexpression of Dp110D954A reduces adult wing and eye size. Thus, imaginal disc growth might be controlled by a mechanism that involves Dp110 and p60 (Weinkove, 1999).

To test this hypothesis using experiments directly comparable to the cell-cycle experiments described above, the effect of En- GAL4-driven Dp110 or Dp110D954A expression on the growth of the posterior compartment of the wing imaginal disc was examined. The size of the posterior compartment is significantly increased by Dp110 expression and reduced by Dp110D954A expression. In contrast, the size of the anterior compartment for each genotype is not altered significantly. In addition, when the ratio of anterior to posterior compartment size was calculated for each disc, it was found that Dp110 expression consistently reduces the ratio, whereas Dp110D954A expression consistently increases the ratio. Thus, unlike direct manipulation of the cell cycle, manipulation of activity of Dp110 is sufficient to alter relative compartment size. Together, the experiments described demonstrate that Dp110 modulates both cell size and cell number, and that manipulation of Dp110 activity can override the intrinsic control mechanisms that normally regulate imaginal disc growth and maintain consistent compartment and organ size (Weinkove, 1999).

An important question arising from this work is how does signaling through Dp110 elicit changes at the cellular level that promote growth? Insulin-stimulated mammalian class IA PI 3-kinase activation regulates various processes that influence growth, including glycogen synthesis, glucose uptake and the translation of mRNAs with 5' oligopyrimidine tracts. Furthermore, numerous reports have shown that class IA PI 3-kinases can regulate cell number through effects on cell proliferation and cell survival. Various experiments suggest that manipulating cell division, cell survival or protein synthesis in isolation does not modulate disc size. In contrast, when cell division is induced (by coexpression of cell cycle regulators, and apoptosis is simultaneously inhibited (by co-expression of p35), imaginal disc compartment size is increased. This is accompanied by an extended larval period, which would allow increased biosynthesis, including protein synthesis, to occur. Thus, it is possible that class IA PI 3-kinase activity can modulate organ size because it simultaneously influences cell division, cell survival and biosynthesis (Weinkove, 1999).

Pten, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell size, cell number, and organ size. In mosaic animals, Pten minus cells proliferate faster than their heterozygous siblings, show an autonomous increase in cell size, and form organs of increased size, whereas overexpression of Pten results in opposite phenotypes. The loss-of-function phenotypes of Pten are suppressed by mutations in the PI3K target Dakt1 and the translational initiation factor eif4A, suggesting that Pten acts through the PI3K signaling pathway to regulate translation. Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of Pten stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and -independent pathways. Furthermore, Pten does not play a major role in cell survival during Drosophila development. These results provide a potential explanation for the high frequency of PTEN mutation in human cancer (Gao, 2000).

Genetic interactions between Pten and PI3K, a component of the insulin signaling pathway in Drosophila, were examined. Overexpression of the PI3K catalytic subunit, Dp110, results in increased wing size, while overexpression of a dominant negative Dp110 construct (PI3KDN) results in the opposite phenotype. These phenotypes are due to changes in cell size. Coexpression of Pten with PI3KDN further reduces wing size. In addition, overexpression of Pten suppresses the increased wing size resulting from PI3K overexpression. To further examine the genetic interaction between Pten and PI3K, Pten mutant cells were examined in a genetic background of overexpressing PI3K or PI3KDN. When PI3K is overexpressed, Pten mutant cells are indistinguishable in cell size from their nonmutant siblings, suggesting that overexpression of PI3K results in increased PIP3 levels and increased signaling that cannot be further activated by removing Pten. Conversely, overexpression of PI3KDN partially suppresses the increased cell size of Pten mutant cells. Thus, PTEN and PI3K antagonize each other in regulating cell size (Gao, 2000).

Certain combinations of loss-of-function alleles of the Drosophila insulin receptor (inr) result in flies with a decreased cell size. This provides an opportunity to examine the genetic epistasis between Pten and inr. The increased cell size of Pten mutant cells can not be reversed in inr mutant animals, and thus loss of Pten function is epistatic to (acts downstream from) mutations in inr (Gao, 2000).

Previous studies have shown that loss of PTEN function promotes cell survival in mammals through activation of Akt. In addition, PTEN acts through Akt in metabolic and longevity control in C. elegans. Dakt1, a Drosophila homolog of Akt, has been suggested to play a role in cell survival in embryogenesis (Staveley, 1998) and cell size control (Verdu, 1999). A hypomorphic allele of Dakt1 has been identified in the large-scale gene disruption project carried out by the Berkeley Drosophila Genome Project. This allele is semilethal, and homozygous survivors show reduced body size and cell size, consistent with a role of Dakt1 in growth control. To examine whether Pten controls cell size through regulating Akt activity, Pten mutant clones were generated in Dakt1 mutant animals. Dakt1 mutation completely suppressed the increase of cell size associated with the Pten mutation. This result provides strong in vivo evidence that Dakt1 functions downstream of (or in parallel to) Pten in the control of cell size. Taken together, these genetic interactions suggest that the role of Pten in opposing signaling through the PI3K/Akt pathway is conserved between flies and vertebrates (Gao, 2000).

It is proposed that Pten regulates cell proliferation by multiple mechanisms, both PI3K-dependent and -independent. One potential PI3K-independent mechanism is suggested by the domain in Pten related to tensin, an actin filament capping protein that localizes to focal adhesions. Overexpression of tensin can suppress anchorage-independent proliferation of Ras-transformed 3T3 cells, and therefore this domain may provide a growth-regulatory function in mammalian PTEN as well. Moreover, in addition to its role as a lipid phosphatase, PTEN also possesses a dual-specificity protein phosphatase activity. PTEN has been shown to bind and dephosphorylate the focal adhesion kinase FAK and to down-regulate the formation of focal adhesions. Such cell contacts play a critical role in regulating proliferation in Drosophila, and the gene products of several Drosophila tumor suppressors such as expanded, fat, and l(2) discs large all localize to adherens or septate junctions. The results for Pten are thus consistent with a model in which PTEN suppresses cell growth and G1/S progression by down-regulating the PI3K/Akt pathway and inhibiting the G2/M transition through an alternative mechanism, perhaps involving regulation of the cytoarchitecture. The ability to regulate both growth and cell division may explain why PTEN is such a common target in advanced tumors. This model is also consistent with the different mutant phenotypes between a null Pten allele and an allele that carries a point mutation (Huang, 1999). While the point mutation changes an invariant amino acid within the phosphatase active site and is likely to inactivate the lipid phosphatase activity, the other domains of Pten are still intact. Characterization of Pten mutants that are specifically defective in cell growth or proliferation may shed further light on its role in the control of overall growth (Gao, 2000).

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

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

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

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

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

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

Adult cell size is measured by the exposed planar surface of the cuticle cells. In principle, this may not reflect the size of the proliferating cells, when organ size is determined. However, in some of the cases examined in this study, cell dissociation has revealed by direct estimation the larger or reduced cell size in the proliferating wing disc cells. In others, cell size during growth is inferred by the mutant effects on pattern formation, a process that precedes final cell differentiation, as in the notum pattern of microchaetae. This pattern results from the singularization of sensory organ mother cells (SOMC) in a field of epidermal cells through a process of lateral inhibition in a field of proneural clusters. Thus, the final pattern reveals cell-cell interactions or communication, as observed in the form of cell projections emanating from epidermal cells. It holds for all mutant and genetic combinations examined in this study that the pattern, number and density of chaetae are all altered in the notum (in the mutant Dmcdc2E1-24 cells fail to differentiate chaetae). In all cases, chaetae appear more densely spaced (separated by less epidermal cells) associated with an increase in the total number of chaetae. These variations to the wild-tupe condition suggest that mutant cells have impaired the capacity to signal among themselves to define spaced SOMC singularization. Whether this is or is not associated with cell size in individual cases is not known. These pattern effects reveal abnormal cell communication between cells during cell proliferation (Resino, 2004).

Although less easy to measure in mosaic nota, there is a phenotypic association of variable cell size with a reduction (in l(3)Me10, gigMe109, Dp110D945A) or an increase (EP(3)3622, fta13, Dp110-CAAX) in notum sizes. But there is no apparent causal relation between both parameters of cell size and number of cells making the adult notum. Perhaps cell viability associated with the mutation, as in l(3)Me10 and gigMe109, may account for the observed lack of correlation between both parameters. However, these effects on notum size in other cases may also reflect failures in cell-cell communication leading to more or less cell proliferation (Resino, 2004).

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

The autonomous effects on reduced clone size can result from the poor viability of mutant cells (l(3)Me10 or Dmcdc2E1-24), as shown in twin clonal analysis and cell death monitoring. The increased clone size of EP(3)3622, fta13 or Dp110-CAAX reflects higher than normal cell proliferation, however there are no correlations between cell size and clone size. Despite this lack of correlation it holds for all mutants examined in this study that, concerning the non-autonomous effects on growth in the mosaic wing sector: the non-mutant cells of the sector are always reduced in number. No cases were found in which the reduction or increase in sector size by the presence of mutant territories is compensated by wild-tupe cells to obtain a normal sized sector (Resino, 2004).

The mosaic wings show, in addition to autonomous effects within mutant sectors, non-autonomous effects in the rest of the wing. It holds for all cases studied that wings with entire or mosaic wing sectors show a reduction in the total area of the wing or more in particular in non-mosaic areas (sectors or compartments) of the wing. This phenomenon is designated as 'positive' or 'negative' accommodation, depending on its correlation with the size of the mutant region. This phenomenon could be easily trivialized for mutations that cause size reduction and 'positive accommodation'. It is arguable that there are not enough cells in the mutant territories to confront with normal growing cells abutting the clone, the sector or the mutant compartment. 'Positive accommodation' could result from adjustment between poorly growing cells and normal ones. However this large effect hardly explain 'negative accommodation' for the whole wing. 'Negative accommodation' occurs in mosaic wings with mutant territories with more cells than normal, such as EP(3)3622, fta13 or Dp110-CAAX (Resino, 2004).

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

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

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

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

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

Modification of ring gland size by ectopic expression of PI3K: Antagonistic actions of ecdysone and insulins determine final size in Drosophila

All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. The larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size (Colombani, 2005).

In metazoans, the insulin/IGF signaling pathway (IIS) plays a key role in regulating growth and metabolism. In Drosophila, a family of insulin-like molecules called Dilps activates a unique insulin receptor (InR) and a conserved downstream kinase cascade that includes PI3-kinase (PI3K) and Akt/PKB. Recent genetic experiments have established that this pathway integrates extrinsic signals such as nutrition with the control of tissue growth during larval stages. The larval period is critical for the control of animal growth, since it establishes the size at which maturation occurs and, consequently, the final adult size. Maturation is itself a complex process that is controlled by the steroid 20-hydroxyecdysone (20E). Peaks of 20E determine the timing of all developmental transitions, from embryo to larva, larva to pupa, and pupa to adult. Ecdysteroids are mainly produced by the prothoracic gland (PG), a part of a composite endocrine tissue called the ring gland. Final adult size thus mainly depends on two parameters: the speed of growth (or growth rate), which is primarily controlled by IIS, and the overall duration of the growth period, which is limited by the onset of the larval-pupal transition and timed by peaks of ecdysone secretion. Very little is known concerning the mechanisms that coordinate these two parameters during larval development (Colombani, 2005).

To investigate the function of ecdysone in controlling organismal growth, a genetic approach was developed that allowed modulation basal levels of ecdysteroids in Drosophila. The initial rationale was to modify the mass of the ring gland in order to change the level of ecdysteroid production. For this goal, the levels of PI3- kinase activity were manipulated in the PG by crossing P0206-Gal4 (P0206>), a line with specific Gal4 expression in the PG and corpora allata (CA), with flies carrying UAS constructs allowing expression of either wild-type (PI3K) or dominant-negative (PI3KDN) PI3-kinase. As expected, these crosses produced dramatic autonomous growth effects in the ring gland, and particularly in the PG: tissue size was increased upon PI3K activation and decreased upon inhibition. Surprisingly, the changes in ring gland growth were accompanied by opposite effects at the organismal level. P0206>PI3K animals (with large ring glands) showed reduced growth at all stages of development and produced emerging adults with reduced size and body weight (78% of wt). Conversely, reducing PI3K activity in the ring gland of P0206>PI3KDN animals led to increased growth and produced adults with 17% greater weight on average. Adult size increase was attributable to an increase in cell number in the wing and the eye. Adult size reduction was accompanied by a decrease in cell number in the wing and in cell size in the eye (Colombani, 2005).

Importantly, the timing of embryonic and larval development of these animals was comparable to control. Both the L2 to L3 transition as well as the cessation of feeding (wandering) occurred at identical times. Further, animals entered pupal development at the same time, except for P0206>PI3KDN animals, which showed a 1-2 hrs delay intrinsic to the UAS-PI3KDN line itself. The duration of pupal development was slightly modified, however, as adult emergence was delayed in P0206>PI3K animals and advanced in P0206>PI3KDN animals, albeit by less than 4 hours following 10 days of development. In contrast, the speed of larval growth was found to be increased in P0206>PI3KDN animals and decreased in the P0206>PI3K animals background at the earliest stage that could be measured (early L2 instar). Because none of these effects were observed when PI3K levels were modified specifically in the CA using the Aug21- Gal4 driver, it was concluded that the observed phenotypes are solely due to PI3K modulation in the PG. Together, these results demonstrate that manipulating PI3K levels in the PG induces non-autonomous changes in the speed of larval growth (growth rate effects), without changing the timing of larval development (Colombani, 2005).

To investigate whether these effects could be attributed to changes in 20E levels, ecdysteroid titers were measured in third instar larvae of the different genotypes. Early after ecdysis into third instar (74hrs AED) ecdysteroids are present at basal level. They accumulate to an intermediate plateau around 90hrs AED and reach peak levels before pupariation (120hrs AED). Because early L3 levels are below the detection limit of the EIA assay, ecdysteroid titers were measured at the intermediate plateau (90hrs AED). In these conditions, a very modest increase of ecdysteroids was observed in P0206>PI3K animals larvae and a small but significant decrease in P0206>PI3KDN animals animals. This was further confirmed by measuring the transcript levels of a direct target of 20E, E74B, which responds to low/moderate levels of 20E. However, in early L3 larvae with basal ecdysteroid levels (74hrs AED), differences in E74B transcripts were clearly visible, with a 1.9-fold increase seen for P0206>PI3K animals and a 1.7-fold decrease for P0206>PI3KDN animals. This establishes that basal circulating levels of 20E are modified in response to manipulation of PI3K levels in the PG. It also suggests that the differences observed on basal 20E level off with the strong global increase of ecdysteroids in mid/late L3 (Colombani, 2005).

Several related lines of evidence strengthen these results: (1) the increase in growth rate and size observed in P0206>PI3KDN animals can be efficiently reverted by adding 20E to their food; (2) feeding wild-type larvae 20E recapitulates the effects observed in P0206>PI3K animals animals; (3) ubiquitous silencing of EcR using an inducible EcR RNAi construct results in a growth increase similar to that observed in P0206>PI3KDN larvae. Finally, the phantom (phm) and disembodied (dib) genes, which are specifically expressed in the PG and encode hydroxylases required for ecdysteroid biosynthesis, showed 1.65- and 2.2- fold increased expression, respectively, upon PI3K activation in the ring gland. This supports the notion that 20E biosynthesis is mildly induced in these experimental conditions. In line with previous results, neither 20E treatment nor EcR silencing has any effect on developmental timing. Overall, the results indicate that manipulating basal levels of 20E signaling in various ways modifies the larval growth rate without affecting the timing of the larval transitions (Colombani, 2005).

Variations in ecdysone levels in animals with different sized ring glands could be due to changes in the PG tissue mass or, alternatively, to a specific effect of PI3K signaling in the secreting tissue. To distinguish between these two possibilities, extra growth was induced in the PG using either dMyc or CyclinD/Cdk4, two potent growth inducers in Drosophila. Although the size of the larval ring gland was markedly increased under these conditions, no effect on pupal or adult size was observed, implying that the size of the ring gland is not the critical factor in the control of body size. Instead, it is likely that the InR/PI3K signaling pathway can specifically activate ecdysone production from the PG (Colombani, 2005).

The possibility was tested that ecdysone signaling opposes the growth-promoting effects of IIS in the larva. To test this, larvae were fed 20E and xPI3K activity was assessed in vivo using a GFP-PH fusion (tGPH) as a marker. Membrane tGPH localization shows a marked decrease in the fat body of 20E-fed animals, and this effect can be reverted by specifically silencing EcR in the fat body. This indicates that ecdysone-induced growth inhibition correlates with decreased IIS, and is mediated through the nuclear receptor EcR. Conversely, larvae with PI3KDN expression in the PG show a 4.2-fold increase in the global levels of dPKB/Akt activity, as measured by the phosphorylation levels of serine 505. In Drosophila cells (as in other metazoan cells) high levels of PI3K/AKT activity cause the transcription factor dFOXO to be retained in the cytoplasm, while low PI3K/AKT activity allows dFOXO to enter the nucleus where it promotes 4E-BP transcription. In larvae with ectopic PI3K expression in the PG, a strong increase is observed in nuclear dFOXO in fat body cells. Similar results were obtained by feeding larvae with 20E. Conversely, inactivation of EcR signaling in fat body cells was carried out using the clonal over-expression of a dominant-negative form of EcR (EcRF645A). In these conditions, a reduction was observed in the accumulation of dFOXO in the nuclei of EcRF645A-expressing cells. As an expected consequence of the increased nuclear dFOXO, global accumulation of 4E-BP transcripts was consistently higher in P0206>PI3K animals as well as in 20E-fed early L3 larvae as compared to control animals, and reduced in arm>EcR-RNAi animals. Together, these results indicate that ecdysone-dependent inhibition of larval growth correlates with a general alteration of insulin/IGF signaling, and a relocalization of dFOXO into the cell nuclei. To more directly test the role of dFOXO in the growth-inhibitory function of ecdysone signaling, the effects of increasing ecdysone levels were examined in a dFOXOmutant genetic background. Although homozygous dFOXO21 animals do not display a detectable growth phenotype, introducing the dFOXO21 mutation was sufficient to totally revert the growth defects of P0206>PI3K animals animals, either when homozygous or heterozygous. This data establishes that dFOXO is required for 20E-mediated growth inhibition (Colombani, 2005).

The endocrine activities of the brain and the fat body have previously been implicated in the humoral control of larval growth. In order to test for possible roles of these two organs in mediating the systemic growth effects of ecdysone, EcR expression was silenced specifically in the brain cells that produce insulins (IPCs) or in the fat body. While specific expression of EcR RNAi in the IPCs fails to reproduce the overgrowth observed in armGal4>EcR-RNAi animals, EcR silencing in the fat body elicits an acceleration of larval growth and a remarkable increase in pupal size. Moreover, no detectable delay in the larval timing was observed in pplGal4>EcR-RNAi animals. Thus, specifically reducing 20E signaling in the fat body is sufficient to recapitulate the systemic effects of global EcR silencing. This demonstrates that the fat body is a major target for ecdysone, and that this tissue can act to relay the 20E growth-inhibitory signal to all larval tissues (Colombani, 2005).

In summary, these results establish an additional role for 20E in modulating animal growth rates. This function is mediated by an antagonistic interaction with IIS that ultimately targets dFOXO function. A similar antagonistic interaction between 20E and insulin signaling controls developmentally-regulated autophagy in Drosophila larva (Colombani, 2005).

Although a direct effect of ecdysone on the cellular growth rate of all larval tissues cannot be ruled out, the experiments reveal a key role for the fat body in relaying ecdysone-dependent growth control signals. Together with previous work, these data suggest that various inputs such as nutrition and ecdysone converge on this important regulatory organ, which then controls the general IIS to modulate organismal growth (Colombani, 2005).

How then is growth connected to developmental timing? The finding that 20E can modulate growth rates in addition to developmental transitions places this hormone in a central position for coordinating these two key processes and controlling organismal size (Colombani, 2005).

Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase

A link exists between natural variation in a Drosophila melanogaster overwintering strategy, diapause, to the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene, Dp110. Variation in diapause, a reproductive arrest, was associated with Dp110 by using Dp110 deletions and genomic rescue fragments in transgenic flies. Deletions of Dp110 increased the proportion of individuals in diapause, whereas expression of Dp110 in the nervous system, but not including the visual system, decreased it. The roles of phosphatidylinositol 3-kinase for both diapause in D. melanogaster and dauer formation in Caenorhabditis elegans suggest a conserved role for this kinase in both reproductive and developmental arrests in response to environmental stresses (Williams, 2006).

Little is known about genes that harbor ecologically relevant allelic variation in natural populations or the degree of conservation of such variation across species. Arrests in development are widespread. For example, mammals hibernate, insects enter diapause, and nematodes form dauer larvae all in response to adverse conditions. Caenorhabditis elegans arrests development to form dauer larvae in response to harsh environmental conditions such as food depletion and overcrowding; dauer larvae have decreased metabolism and increased fat storage. When confronted with low temperature and short days (SDs), Drosophila melanogaster enters an ovarian reproductive diapause where adult females have immature ovaries and exclusively previtellogenic oocytes. It is not known whether common mechanisms underlie these arrests because, although much is known about the genetic underpinnings of dauer formation in C. elegans, little is known about genes involved in reproductive diapause (Williams, 2006).

Ecological studies show that adult reproductive diapause is a powerful overwintering strategy for many insects including Drosophila. Diapause is advantageous in northern climates because it enables females to survive for several months through harsh winter conditions and then emerge and lay eggs when temperatures increase and days get longer. Arrests in response to adverse conditions are of interest because they can provide an evolutionary framework in which to interpret existing genetic variation both within and between species. Diapause is associated with resource allocation trade-offs involved in life history strategies, for example, whether to allocate resources to growth and survivorship or reproduction. D. melanogaster lines that vary in latitudinal origin differ in the proportion of individuals in diapause as well as in a suite of fitness-related traits that are genetically correlated to diapause. These traits include life span, age-specific mortality, fecundity, resistance to cold and starvation stress, lipid content, development time, and egg-to-adult viability. This study asked what gene(s) or gene pathways play a role in natural variation in diapause and potentially underlie this pleiotropy (Williams, 2006).

Two natural diapause variants of D. melanogaster were used as a basis to identify genetic regulators of diapause. The Windsor (W) natural variant from Canada exhibits an autosomal-recessive high-diapause phenotype compared with the Cartersville (C) natural variant from the Southern U.S., which confers a fully dominant low-diapause phenotype. Crosses between the variants suggested that diapause may be influenced by relatively few genes (Williams, 2006).

This study is consistent with a model in which reducing signaling via the insulin/PI3-kinase pathway increases D. melanogaster diapause. The finding that altered PI3-kinase expression in neurons alone can affect fly diapause is reminiscent of the observation that expression of PI3-kinase in C. elegans neurons can restore dauer formation to wild-type levels in age-1 (PI3-kinase) null mutant worms. Furthermore, mutations that lower nematode insulin signaling promote dauer larval formation suggesting the exciting possibility of a shared mechanism for developmental and reproductive arrests in D. melanogaster ovarian diapause and nematode dauer formation between these species' distant relatives. The results suggest that insulin-signaling genes known to play important roles in dauer formation in C. elegans are excellent candidate genes for further investigation into the mechanisms involved in diapause in D. melangaster and the many other species that undergo developmental and reproductive arrests (Williams, 2006).

Various fitness-related phenotypes are genetically correlated to D. melanogaster diapause in nature. This genetic correlation could be explained by pleiotropic effects of the Dp110 gene. Notably, mutational analyses of the insulin-regulated PI3-kinase and other genes in the insulin-signaling pathway in D. melanogaster have revealed affects on lifespan, development, body size and growth, nutrient stress, lipid content, locomotor activity, and egg chamber development. The findings suggest that Dp110 is not only an important regulator of diapause in D. melanogaster but also, through its pleiotropic effects, may influence the suite of life-history trade-offs associated with diapause in natural populations (Williams, 2006).

Few genes are known to affect reproductive diapause. The discovery of a role for Dp110 in reproductive diapause in D. melanogaster is significant because this species is amenable to mutational analyses, transgenic manipulations, and genomic investigations of the various phases of diapause including its photoperiodic induction, maintenance, and termination. Additionally, once genes that affect diapause have been identified, they can be used as candidate genes for the investigation of diapause in other species. From an evolutionary perspective, diapause is an important adaptive trait linked to many life-history parameters important for survivorship; the identification of genes involved in diapause is an entry point into studies of the molecular evolution of these genes. Finally, from an applied perspective, genetic manipulations of diapause can be engineered to control pest species and maximize the efficiency of their biological control agents (Williams, 2006).

Diapause is an adaptive trait critical for survival in temperate climates. The identification of a single major gene regulator of diapause in nature provides a tantalizing example of the small but growing body of literature that reveals that major adaptive phenotypes are affected by variation in genes with large effects (single genes). This finding is in conflict with the historically prominent notion of Fisher, Haldane, and Wright who envisioned hundreds of interactive genes, each having tiny additive effects on a trait. These genes by definition could not be localized or identified because their effect sizes were so small. However, contrary to this model, recent data suggest that genes can have large effects on adaptive phenotypes and that they exist along with genes with small effects. Challenges for the future are to identify both large and smaller effect genes and understand how they interact with the environment to generate variation in traits of adaptive significance (Williams, 2006).

Synergism between altered cortical polarity and the PI3K/TOR pathway in the suppression of tumour growth

Loss of function of pins (partner of inscuteable) partially disrupts neuroblast (NB) polarity and asymmetric division, results in fewer and smaller NBs and inhibits Drosophila larval brain growth. Food deprivation also inhibits growth. However, this study found that the combination of loss of function of pins and dietary restriction results in loss of NB asymmetry, overproliferation of Miranda-expressing cells, brain overgrowth and increased frequency of tumour growth on allograft transplantation. The same effects are observed in well-fed pins larvae that are mutant for pi3k (phosphatidylinositol 3-kinase) or exposed to the TOR inhibitor rapamycin. Thus, pathways that are sensitive to food deprivation and dependent on PI3K and TOR are essential to suppress tumour growth in Drosophila larval brains with compromised pins function. These results highlight an unexpected crosstalk whereby the normally growth-promoting, nutrient-sensing PI3K/TOR pathway suppresses tumour formation in neural stem cells with compromised cell polarity (Rossi, 2012).

PI3K and TOR activity are necessary for sustained growth and proliferation, and to resume cycling activity after developmentally programmed quiescence. Consistently, there is abundant experimental evidence showing that in most tumour types growth is coupled to activated PI3K, and not to its loss. Evidence showing that low-calorie intake is a protective condition against malignant transformation is also abundant. However, the current results indicate that in Drosophila PI3K and TOR activity and a normal food supply have a function in preventing overgrowth in neural stem cells with compromised cortical polarity. Whether this conclusion applies to vertebrates remains to be ascertained. Interestingly, some PI3Kγ−/− mouse strains have a high incidence of invasive colorectal tumours, which is not due to PI3Kγ loss alone, but is suspected to result from the combined effect of PI3Kγ loss and other unknown factors. Taking into account the fact that the pathways involved in the control of cell polarity and in the response to changing nutrient conditions are largely conserved across species, it is proposed that similar synergistic interactions might take place in vertebrates (Rossi, 2012).


Albert, S., et al. (1997). Isolation and characterization of the droPIK57 gene encoding a new regulatory subunit of phosphatidylinositol 3-kinase from Drosophila melanogaster. Gene 198(1-2): 181-9. PubMed Citation: 9370280

Anderson, K. E., et al. (2000). DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation. Curr. Biol. 10: 1403-1412. 11102801

Antonetti, D. A., Algenstaedt, P., Kahn, C. R. (1996). Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain. Mol. Cell. Biol. 16(5): 2195-203. PubMed Citation: 8628286

Araki, E., et al. (1994). Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372(6502): 186-90. PubMed Citation: 7526222

Arendt, K. L., Royo, M., Fernandez-Monreal, M., Knafo, S., Petrok, C. N., Martens, J. R. and Esteban, J. A. (2010). PIP3 controls synaptic function by maintaining AMPA receptor clustering at the postsynaptic membrane. Nat Neurosci 13: 36-44. PubMed ID: 20010819

Atwall, J. K., et al. (2000). The TrkB-Shc site signals neuronal survival and local axon growth via MEK and PI3-kinase. Neuron 27: 265-277. PubMed Citation: 10985347

Attwell, S., Mills, J., Troussard, A., Wu, C., Dedhar, S. (2003). Integration of cell attachment, cytoskeletal localization, and signaling by integrin-linked kinase (ILK), CH-ILKBP, and the tumor suppressor PTEN. Mol. Biol. Cell. 14(12): 4813-25. 12960424

Barrett, A.L., Krueger, S., and Datta, S. (2008). Branchless and Hedgehog operate in a positive feedback loop to regulate the initiation of neuroblast division in the Drosophila larval brain. Dev. Biol. 317: 234-245. PubMed Citation: 18353301

Bartlett, S. E., et al. (1999). Differential mRNA expression and subcellular locations of PI3-kinase isoforms in sympathetic and sensory neurons. J. Neurosci. Res. 56(1): 44-53. PubMed Citation: 10213474

Bernal, A. and Kimbrell, D. A. (2000). Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity. Proc. Natl. Acad. Sci. 97(11): 6019-6024. PubMed Citation: 10811906

Berry, D. L. and Baehrecke, E. H. (2007). Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila. Cell 131(6): 1137-1148. PubMed Citation: 18083103

Bi, L., et al. (1999). Proliferative defect and embryonic lethality in mice homozygous for a deletion in the p110alpha subunit of phosphoinositide 3-kinase. J. Biol. Chem. 274(16): 10963-8

Biggs, W. H., et al. (1999). Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc. Natl. Acad. Sci. 96(13): 7421-6. PubMed Citation: 10377430

Bohni, R., et al. (1999). Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4. Cell 97(7): 865-75. PubMed Citation: 10399915

Brennan, P., et al. (1999). p70(s6k) integrates phosphatidylinositol 3-kinase and rapamycin-regulated signals for E2F regulation in T lymphocytes. Mol. Cell. Biol. 19(7): 4729-38. PubMed Citation: 10373522

Britton, J. S. and Edgar, B. A. (1998). Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. Development 125(11): 2149-58. 9570778

Britton, J. S., et al. (2002). Drosophila's insulin/pi3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2: 239-249. 11832249

Brogiolo, W., et al. (2001). An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control. Curr. Biol. 11: 213-221. 11250149

Caldwell, P.E., Walkiewicz, M., and Stern, M. (2005). Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release. Curr. Biol. 15: 1785-1795. Medline abstract: 16182526

Campana, W. M., Darin, S. J. and O'Brien, J. S. (1999). Phosphatidylinositol 3-kinase and akt protein kinase mediate IGF-I- and prosaptide-induced survival in schwann cells. J. Neurosci. Res. 57(3): 332-41. PubMed Citation: 10412024

Carballada, R., Yasuo, H. and Lemaire, P. (2001). Phosphatidylinositol-3 kinase acts in parallel to the ERK MAP kinase in the FGF pathway during Xenopus mesoderm induction. Development 128: 35-44. 11092809

Chalhoub, N., Zhu, G., Zhu, X. and Baker, S. J. (2009). Cell type specificity of PI3K signaling in Pdk1- and Pten-deficient brains. Genes Dev. 23(14): 1619-24. PubMed Citation: 19605683

Chang, C., et al. (2006). MIG-10/lamellipodin and AGE-1/PI3K promote axon guidance and outgrowth in response to slit and netrin. Curr. Biol. 16(9): 854-62. 16618541

Chaudhary, A., et al. (2000). Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338. Curr. Biol. 10: 551-554. PubMed Citation: 10801448

Chaves, I., van der Horst, G. T., Schellevis, R., Nijman, R. M., Koerkamp, M. G., Holstege, F. C., Smidt, M. P. and Hoekman, M. F. (2014). Insulin-FOXO3 signaling modulates circadian rhythms via regulation of clock transcription. Curr Biol 24: 1248-1255. PubMed ID: 24856209

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

Chen, H. C. and Guan, J. L. (1994). Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. 91(21): 10148-52. PubMed Citation: 7937853

Chen, L., et al. (2007). PLA2 and PI3K/PTEN pathways act in parallel to mediate chemotaxis. Dev. Cell 12(4): 603-14. Medline abstract: 17419997

Cheng, L. Y., Bailey, A. P., Leevers, S. J., Ragan, T. J., Driscoll, P. C. and Gould, A. P. (2011). Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila. Cell 146: 435-447. PubMed ID: 21816278

Cho, K. S., et al. (2001). Drosophila phosphoinositide-dependent kinase-1 regulates apoptosis and growth via the phosphoinositide 3-kinase-dependent signaling pathway. Proc. Natl. Acad. Sci. 98: 6144-6149. 11344272

Chung, C. Y., Potikyan, G. and Firtel, R. A. (2001). Control of cell polarity and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa. Mol. Cell 7: 937-947. 11389841

Colombani, J., et al. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114: 739-749. PubMed Citation: 14505573

Colombani, J., et al. (2005). Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science 310(5748): 667-70. 16179433

Crackower, M. A., et al. (2002). Regulation of myocardial contractility and cell size by distinct PI3K-PTEN signaling pathways. Cell 110: 737-749. 12297047

Cuesto, G., Enriquez-Barreto, L., Carames, C., Cantarero, M., Gasull, X., Sandi, C., Ferrus, A., Acebes, A. and Morales, M. (2011). Phosphoinositide-3-kinase activation controls synaptogenesis and spinogenesis in hippocampal neurons. J Neurosci 31: 2721-2733. PubMed ID: 21414895

Dan, H. C., et al. (2002). Phosphatidylinositol 3-kinase/Akt pathway regulates tuberous sclerosis tumor suppressor complex by phosphorylation of tuberin. J. Biol. Chem. 277: 35364-35370. 12167664

Dennis, P., et al. (2001). Mammalian TOR: a homeostatic ATP sensor. Science 294: 1102-1105. 11691993

Dhand, R., et al (1994). PI 3-kinase: structural and functional analysis of intersubunit interactions. EMBO J. 13(3): 511-21. PubMed ID: 8313896

Diaz-Guerra, M. J., et al. (1999). Negative regulation by phosphatidylinositol 3-kinase of inducible nitric oxide synthase expression in macrophages. J. Immunol. 162(10): 6184-90. PubMed ID: 10229863

Dormann, D., et al. (2002). Visualizing PI3 kinase-mediated cell-cell signaling during Dictyostelium development. Curr. Biol. 12: 1178-1188. 12176327

Dufner, A., et al. (1999). Protein kinase B localization and activation differentially affect S6 kinase 1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation. Mol. Cell. Biol. 19(6): 4525-34. PubMed ID: 10330191

Dumstrei, K., Wang, F., and Hartenstein, V. (2003). Role of DE-cadherin in neuroblast proliferation, neural morphogenesis, and axon tract formation in Drosophila larval brain development. J. Neurosci. 23: 3325-3335. PubMed Citation: 12716940

Dutta, S. and Baehrecke, E. H. (2008). Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila. Curr. Biol. 18(19): 1466-75. PubMed Citation: 18818081

Egawa, K., et al. (1999). Membrane-targeted phosphatidylinositol 3-kinase mimics insulin actions and induces a state of cellular insulin resistance. J. Biol. Chem. 274(20): 14306-14. PubMed ID: 10318852

Fantauzzo, K. A. and Soriano, P. (2014). PI3K-mediated PDGFRalpha signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways. Genes Dev 28: 1005-1017. PubMed ID: 24788519

Porstmann, T., et al. (2008). SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth. Cell Metab. 8(3): 224-36. PubMed Citation: 18762023

Frese, K. K., et al. (2006). Oncogenic function for the Dlg1 mammalian homolog of the Drosophila discs-large tumor suppressor. EMBO J. 25(6): 1406-17. 16511562

Fuller, C. L., Ravichandran, K. S. and Braciale, V. L. (1999). Phosphatidylinositol 3-kinase-dependent and -independent cytolytic effector functions. J. Immunol. 162(11): 6337-40. PubMed ID: 10352245

Funamoto, S., Meili, R., Lee, S., Parry, L. and Firtel, R. A. (2002). Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell 109: 611-623. 12062104

Fuss, B., Becker, T., Zinke, I. and Hoch, M. (2006). The cytohesin Steppke is essential for insulin signalling in Drosophila. Nature 444: 945-948. PubMed Citation: 17167488

Galloni, M., and Edgar, B. A. (1999). Cell-autonomous and non-autonomous growth-defective mutants of Drosophila melanogaster. Development 126: 2365-2375. PubMed ID: 10225996

Gao, X., Neufeld, T. P. Pan, D. (2000). Drosophila PTEN regulates cell growth and proliferation through PI3K-dependent and -independent pathways. Dev. Biol. 221: 404-418. PubMed ID: 10790335

Gassama-Diagne, A., et al. (2006). Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells. Nat. Cell Biol. 8(9): 963-70. Medline abstract: 16921364

Gautreau, A., et al. (1999). Ezrin, a plasma membrane-microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway. Proc. Natl. Acad. Sci. 96(13): 7300-5. PubMed ID: 10377409

Giglione, C. and Parmeggiani, A. (1998). Raf-1 is involved in the regulation of the interaction between guanine nucleotide exchange factor and Ha-ras. Evidences for a function of Raf-1 and phosphatidylinositol 3-kinase upstream to Ras. J. Biol. Chem. 273(52): 34737-44. PubMed ID: 9856997

Gille, H. and Downward, J. (1999). Multiple ras effector pathways contribute to G(1) cell cycle progression. J. Biol. Chem. 274(31): 22033-40. PubMed ID: 10419529

Godbout, J. P., et al. (1999). Insulin activates caspase-3 by a phosphatidylinositol 3'-kinase-dependent pathway. Cell. Signal. 11(1): 15-23. PubMed ID: 10206340

Goberdhan, D. C., et al. (1999). Drosophila tumor suppressor PTEN controls cell size and number by antagonizing the Chico/PI3-kinase signaling pathway. Genes Dev. 13(24): 3244-58. PubMed ID: 10617573

Greenwood, J. A., et al. (2000). Restructuring of focal adhesion plaques by PI 3-kinase. Regulation by PtdIns (3,4,5)-p3 binding to alpha-actinin. J. Cell Biol. 150(3): 627-42. 10931873

Grumont, R. J., Strasser, A. and Gerondakis, S. (2003). B cell growth is controlled by Phosphatidylinosotol 3-kinase-dependent induction of Rel/NF-kappaB regulated c-myc transcription. Mol. Cell 10: 1283-1294. 12504005

Gupta, S., et al. (2007). Binding of ras to phosphoinositide 3-kinase p110alpha is required for ras-driven tumorigenesis in mice. Cell 129(5): 957-68. PubMed citation: 17540175

Hamada, K., et al. (2005). The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis. Genes Dev. 19: 2054-2065. 16107612

Hanna, A. N., et al. (1999). A novel pathway for tumor necrosis factor-alpha and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3-kinase. J. Biol. Chem. 274(18): 12722-9

Hanna, N., et al. (2006). Reduced phosphatase activity of SHP-2 in LEOPARD syndrome: Consequences for PI3K binding on Gab1. FEBS Lett. 580(10): 2477-2482. 16638574

Harpur, A. G., et al. (1999). Intermolecular interactions of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase. J. Biol. Chem. 274(18): 12323-32

Hart, K. C., Robertson, S. C. and Donoghue, D. J. (2001). Identification of tyrosine residues in constitutively activated Fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and Phosphatidylinositol 3-kinase activation. Mol. Biol. Cell 12: 931-942. 11294897

Hopper, N. A. (2006). The adaptor protein soc-1/Gab1 modifies growth factor receptor output in C. elegans. Genetics 173(1): 163-75. 16547100

Hsu, H. J. and Drummond-Barbosa, D. (2009). Insulin levels control female germline stem cell maintenance via the niche in Drosophila, Proc. Natl Acad. Sci. 106: 1117-1121. PubMed Citation: 19136634

Hsu, H. J. and Drummond-Barbosa, D. (2011). Insulin signals control the competence of the Drosophila female germline stem cell niche to respond to Notch ligands. Dev. Biol. 350(2): 290-300. PubMed Citation: 21145317

Huang, C. H., et al. (2007). The structure of a human p110α/p85α complex elucidates the effects of oncogenic PI3Kα mutations. Science 318(5857): 1744-8. PubMed citation; Online text

Huang, H., et al. (1999). PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development. Development 126(23): 5365-72. PubMed Citation: 10556061

Hyun, S., et al. (2009). Conserved MicroRNA miR-8/miR-200 and its target USH/FOG2 control growth by regulating PI3K. Cell 139(6): 1096-108. PubMed Citation: 20005803

Iiboshi, Y., et al. (1999). Amino acid-dependent control of p70(s6k). Involvement of tRNA aminoacylation in the regulation. J. Biol. Chem. 274: 1092-1099. 9873056

Innocenti, M., et al. (2003). Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1. J. Cell Biol. 160: 17-23. Medline abstract: 12515821

Inukai, K., et al. (1997). p85alpha gene generates three isoforms of regulatory subunit for phosphatidylinositol 3-kinase (PI 3-Kinase), p50alpha, p55alpha, and p85alpha, with different PI 3-kinase activity elevating responses to insulin. J. Biol. Chem. 272(12): 7873-82

Isakoff, S. J., et al. (1998). Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast. EMBO J. 17(18): 5374-87

Jiang, B. H., et al. (2001). Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell Growth Differ. 12(7): 363-9. 11457733

Johnson, C., Chun-Jen Lin, C. and Stern, M. (2012). Ras-dependent and Ras-independent effects of PI3K in Drosophila motor neurons. Genes Brain Behav. 11(7): 848-58. PubMed Citation: 22783951

Jones, S. M., et al. (1999). PDGF induces an early and a late wave of PI 3-kinase activity, and only the late wave is required for progression through G1. Curr. Biol. 9(10): 512-21

Jordan-Alvarez, S., Fouquet, W., Sigrist, S. J. and Acebes, A. (2012). Presynaptic PI3K activity triggers the formation of glutamate receptors at neuromuscular terminals of Drosophila. J Cell Sci 125: 3621-3629. PubMed ID: 22505608

Kaliman, P., et al. (1999). Insulin-like growth factor-II, phosphatidylinositol 3-kinase, nuclear factor-kappaB and inducible nitric-oxide synthase define a common myogenic signaling pathway. J. Biol. Chem. 274(25): 17437-44

Kenney, A. M., Widlund, H. R. and Rowitch, D. H. (2004). Hedgehog and PI-3 kinase signaling converge on Nmyc1 to promote cell cycle progression in cerebellar neuronal precursors. Development 131: 217-228 . 14660435

Kingham, E. and Welham, M. (2009). Distinct roles for isoforms of the catalytic subunit of class-IA PI3K in the regulation of behaviour of murine embryonic stem cells. J. Cell Sci. 122(Pt 13): 2311-21. PubMed Citation: 19509054

Kleijn, M. and Proud, C. G. (2000). Glucose and amino acids modulate translation factor activation by growth factors in PC12 cells. Biochem. J. 347: 399-406. 10749669

Klesse, L. J. and Parada, L. F. (1998). p21 ras and phosphatidylinositol-3 kinase are required for survival of wild-type and NF1 mutant sensory neurons. J. Neurosci. 18(24): 10420-8

Klesse, L. J., et al. (1999). Nerve growth factor induces survival and differentiation through two distinct signaling cascades in PC12 cells. Oncogene 18(12): 2055-68

Komada, M. and Soriano, P. (1999). Hrs, a FYVE finger protein localized to early endosomes, is implicated in vesicular traffic and required for ventral folding morphogenesis. Genes Dev. 13(11): 1475-85

Kovacs, E. M., Ali. R. G., McCormack, A. J. and Yap, A. S. (2002). E-cadherin homophilic ligation directly signals through Rac and phosphatidylinositol 3-kinase to regulate adhesive contacts. J. Biol. Chem. 277(8): 6708-18. 11744701

Kubota, Y., et al. (2001). Src transduces erythropoietin-induced differentiation signals through phosphatidylinositol 3-kinase. EMBO J. 20: 5666-5677. 11598010

Kuruvilla, R., Ye, H. and Ginty, D. D. (2000). Spatially and functionally distinct roles of the PI3-K effector pathway during NGF signaling in sympathetic neurons. Neuron 27: 499-512.

Kutateladze, T. G., et al. (1999). Phosphatidylinositol 3-phosphate recognition by the FYVE domain. Mol. Cell 3: 805-811

Kwon, M., et al. (2005). Recruitment of the tyrosine phosphatase SHP-2 to the p85 Subunit of Phosphatidylinositol-3 (PI-3) kinase is required for Insulin-like growth factor I dependent PI-3 kinase activation in smooth muscle cells. Endocrinology 147(3): 1458-65. 16306077

Laprise, P., Viel, A. and Rivard, N (2004). Human homolog of disc-large is required for adherens junction assembly and differentiation of human intestinal epithelial cells. J. Biol. Chem. 279: 10157-10166. 14699157

Larsen, M., et al. (2003). Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis. Dev. Bio. 255: 178-191. 12618142

Lavery, W., et al. (2007). Phosphatidylinositol 3-kinase and Akt nonautonomously promote perineurial glial growth in Drosophila peripheral nerves. J. Neurosci. 27(2): 279-88. PubMed Citation: 17215387

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

Leevers, S. J., et al. (1996). The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth. EMBO J. 15(23): 6584-94

Leverrier, Y., et al. (1999). Role of PI3-kinase in Bcl-X induction and apoptosis inhibition mediated by IL-3 or IGF-1 in Baf-3 cells. Cell Death Differ. 6(3): 290-6

Leverrier, Y. and Ridley, A. J. (2001). Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages. Curr. Biol. 11: 195-199. 11231156

Li, B.-S., et al. (2001). Activation of Phosphatidylinositol-3 kinase (PI-3K) and Extracellular regulated kinases (Erk1/2) is involved in muscarinic receptor-mediated DNA synthesis in neural progenitor cells. J. Neurosci. 21(5): 1569-1579. 11222647

Li, D., et al. (1999). Participation of PI3K and atypical PKC in Na+-K+-pump stimulation by IGF-I in VSMC. Am. J. Physiol. 276(6 Pt 2): H2109-16. PubMed Citation: 10362694

Lin, C. C.-J., Summerville, J. B., Howlett, E. and Stern, M. (2011). The metabotropic glutamate receptor activates the lipid kinase PI3K in Drosophila motor neurons through the calcium/calmodulin-dependent protein kinase II and the nonreceptor tyrosine protein kinase DFak. Genetics 188(3): 601-13. PubMed Citation: 21515581

Lin, C.-H., et al. (2001). A role for the PI-3 kinase signaling pathway in fear conditioning and synaptic plasticity in the amygdala. Neuron 31: 841-851. 11567621

Linassier, C., et al. (1997). Molecular cloning and biochemical characterization of a Drosophila phosphatidylinositol-specific phosphoinositide 3-kinase. Biochem. J. 321 (Pt 3): 849-56

Liu, A. X., et al. (1998). AKT2, a member of the protein kinase B family, is activated by growth factors, v-Ha-ras, and v-src through phosphatidylinositol 3-kinase in human ovarian epithelial cancer cells. Cancer Res. 58(14): 2973-7

Liu, Y. and Lehmann, M. (2006). FOXO-independent suppression of programmed cell death by the PI3K/Akt signaling pathway in Drosophila. Dev. Genes Evol. [Epub ahead of print]. 16520939

Lockyer, P. J., et al. (1999). Identification of the ras GTPase-activating protein GAP1(m) as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo. Curr. Biol. 9(5): 265-8

Lv, W. W., Wei, H. M., Wang, D. L., Ni, J. Q. and Sun, F. L. (2012). Depletion of histone deacetylase 3 antagonizes PI3K-mediated overgrowth through the acetylation of histone H4 at lysine 16. J. Cell Sci. [Epub ahead of print]. PubMed Citation: 22956542

MacDougall, L. K., Domin, J. and Waterfield, M. D. (1995). A family of phosphoinositide 3-kinases in Drosophila identifies a new mediator of signal transduction. Curr. Biol. 5(12): 1404-15

Mairet-Coello, G., Tury, A. and DiCicco-Bloom, E. (2009). Insulin-like growth factor-1 promotes G(1)/S cell cycle progression through bidirectional regulation of cyclins and cyclin-dependent kinase inhibitors via the phosphatidylinositol 3-kinase/Akt pathway in developing rat cerebral cortex. J. Neurosci. 29: 775-788. PubMed Citation: 19158303

Maurange, C., Cheng, L. and Gould, A.P. (2008). Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila. Cell 133: 891-902. PubMed Citation: 18510932

Miled, N., et al. (2007). Mechanism of two classes of cancer mutations in the phosphoinositide 3-kinase catalytic subunit. Science 317(5835): 239-42. PubMed citation; Online text

Man, H. Y., Wang, Q., Lu, W. Y., Ju, W., Ahmadian, G., Liu, L., D'Souza, S., Wong, T. P., Taghibiglou, C., Lu, J., Becker, L. E., Pei, L., Liu, F., Wymann, M. P., MacDonald, J. F. and Wang, Y. T. (2003). Activation of PI3-kinase is required for AMPA receptor insertion during LTP of mEPSCs in cultured hippocampal neurons. Neuron 38: 611-624. PubMed ID: 12765612

Martín-Castellanos, C. and Edgar, B. A. (2002). A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing. Development 129: 1003-1013. 11861483

Martin, D. N., Balgley, B., Dutta, S., Chen, J., Rudnick, P., Cranford, J., Kantartzis, S., DeVoe, D. L., Lee, C. and Baehrecke, E. H. (2007). Proteomic analysis of steroid-triggered autophagic programmed cell death during Drosophila development. Cell Death Differ. 14: 916-923. PubMed Citation: 17256009

Martin-Pena, A., Acebes, A., Rodriguez, J. R., Sorribes, A., de Polavieja, G. G., Fernandez-Funez, P. and Ferrus, A. (2006). Age-independent synaptogenesis by phosphoinositide 3 kinase. J Neurosci 26: 10199-10208. PubMed ID: 17021175

Ming, G., et al. (1999). Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance. Neuron 23(1): 139-48

Minshall, C., et al. (1999). Phosphatidylinositol 3'-kinase, but not S6-kinase, is required for insulin-like growth factor-I and IL-4 to maintain expression of Bcl-2 and promote survival of myeloid progenitors. J. Immunol. 162(8): 4542-9

Miron, M., et al. (2001). The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat. Cell Bio. 3: 596-601. 11389445

Miron, M., Lasko, P. and Sonenberg, N. (2003). Signaling from Akt to FRAP/TOR targets both 4E-BP and S6K in Drosophila melanogaster. Mol. Cell. Biol. 23(24): 9117-26. 14645523

Misra, S., et al. (1999). Phosphoinositide 3-kinase lipid products regulate ATP-dependent transport by sister of P-glycoprotein and multidrug resistance associated protein 2 in bile canalicular membrane vesicles. Proc. Natl. Acad. Sci. 96(10): 5814-9

Moniakis, J., et al. (2001). An SH2-domain-containing kinase negatively regulates the phosphatidylinositol-3 kinase pathway. Genes Dev. 15: 687-698. 11274054

Montagne, J., et al. (1999). Drosophila S6 kinase: a regulator of cell size. Science 285(5436): 2126-9

Montero, J.-A., et al. (2003). Phosphoinositide 3-kinase is required for process outgrowth and cell polarization of gastrulating mesendodermal cells. Curr. Biol. 13: 1279-1289. 12906787

Munday, A. D., et al. (1999). The inositol polyphosphate 4-phosphatase forms a complex with phosphatidylinositol 3-kinase in human platelet cytosol. Proc. Natl. Acad. Sci. 96(7): 3640-5

Nie, S. and Chang, C. (2007). PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation. Mech. Dev. 124(9-10): 657-67. PubMed citation: 17716876

Niewiadomski, P., Kong, J. H., Ahrends, R., Ma, Y., Humke, E. W., Khan, S., Teruel, M. N., Novitch, B. G. and Rohatgi, R. (2014). Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling. Cell Rep 6: 168-181. PubMed ID: 24373970

Nishita, M., et al. (2004). Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase Slingshot and its role for insulin-induced membrane protrusion. J. Biol. Chem. 279(8): 7193-8. 14645219

Oldham, S., et al. (2000). Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14: 2689-2694. 11069885

Ong, S. H., et al. (2001). Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins. Proc. Natl. Acad. Sci. 98(11): 6074-9. 11353842

Orme, M. H. et al. (2006). Input from Ras is required for maximal PI(3)K signalling in Drosophila. Nat. Cell Biol. 8: 1298-1302. PubMed citation: 17041587

Ottinger, E. A., Botfield, M. C. and Shoelson, S. E. (1998). Tandem SH2 domains confer high specificity in tyrosine kinase signaling. J. Biol. Chem. 273(2): 729-35. 9422724

Parisi, F., et al. (2011). Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo. BMC Biol. 9: 65. PubMed Citation: 21951762

Pallard, C., et al. (1999). Distinct roles of the phosphatidylinositol 3-kinase and STAT5 pathways in IL-7-mediated development of human thymocyte precursors. Immunity 10(5): 525-35. PubMed Citation: 10367898

Pastorino, J. G., Tafani, M. and Farber, J. L. (1999). Tumor necrosis factor induces phosphorylation and translocation of BAD through a phosphatidylinositide-3-OH kinase-dependent pathway. J. Biol. Chem. 274(27): 19411-6. PubMed Citation: 10383455

Pece, S., et al. (1999). Activation of the protein kinase Akt/PKB by the formation of E-cadherin-mediated cell-cell junctions. Evidence for the association of phosphatidylinositol 3-kinase with the E-cadherin adhesion complex. J. Biol. Chem. 274(27): 19347-51. PubMed Citation: 10383446

Penuel, E. and Martin, G. S. (1999). Transformation by v-Src: Ras-MAPK and PI3K-mTOR mediate parallel pathways. Mol. Biol. Cell 10(6): 1693-703. PubMed Citation: 10359590

Perkinton, M. S., Sihra, T. S. and Williams, R. J. (1999). Ca2+-permeable AMPA receptors induce phosphorylation of cAMP response element-binding protein through a Phosphatidylinositol 3-kinase-dependent stimulation of the mitogen-activated protein kinase signaling cascade in neurons. J. Neurosci. 19(14): 5861-5874. PubMed Citation: 10407026

Peterson, T. R., et al. (2009). DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 137(5): 873-86. PubMed Citation: 19446321

Piekny, A. J., Wissmann, A. and Mains, P. E. (2000). Embryonic morphogenesis in Caenorhabditis elegans integrates the activity of LET-502 Rho-binding kinase, MEL-11 myosin phosphatase, DAF-2 insulin receptor and FEM-2 PP2c phosphatase. Genetics 156(4): 1671-89. 11102366

Pigazzi, A., et al. (1999). Nitric oxide inhibits thrombin receptor-activating peptide-induced phosphoinositide 3-kinase activity in human platelets. J. Biol. Chem. 274(20): 14368-75. PubMed Citation: 10318860

Pimentel, B., et al. (2002). A role for Phosphoinositide 3-Kinase in the control of cell division and survival during retinal development. Dev. Bio. 247: 295-306. 12086468

Poser, S., et al. (2000). SRF-dependent gene expression is required for PI3-kinase-regulated cell proliferation. EMBO J. 19: 4955-4966. PubMed Citation: 10990459

Potempa, S. and Ridley, A. J. (1998). Activation of both MAP kinase and phosphatidylinositide 3-kinase by Ras is required for hepatocyte growth factor/scatter factor-induced adherens junction disassembly. Mol. Biol. Cell 9(8): 2185-200. PubMed Citation: 9693375

Pritchett, T. L. and McCall, K. (2012). Role of the insulin/Tor signaling network in starvation-induced programmed cell death in Drosophila oogenesis. Cell Death Differ. 19(6): 1069-79. PubMed Citation: 22240900

Prober, D. A. and Edgar, B. A. (2002). Interactions between Ras1, dMyc, and dPI3K signaling in the developing Drosophila wing. Genes Dev. 16: 2286-2299. 12208851

Radke, A. L., et al. (2009). Mature human eosinophils express functional Notch ligands mediating eosinophil autocrine regulation. Blood 113: 3092-3101. PubMed Citation: 19171875

Raught, B., Gingras, A. C. and Sonenberg, N. (2001). The target of rapamycin (TOR) proteins. Proc. Natl. Acad. Sci. 98: 7037-7044. 11416184

Reddy, S. A., Huang, J. H. and Liao, W. S. (1997). Phosphatidylinositol 3-kinase in interleukin 1 signaling. Physical interaction with the interleukin 1 receptor and requirement in NFkappaB and AP-1 activation. J. Biol. Chem. 272(46): 29167-73. PubMed Citation: 9360994

Reiske, H. R., et al. (1999). Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase-promoted cell migration. J. Biol. Chem. 274(18): 12361-6. PubMed Citation: 10212207

Resino, J. and Garcia-Bellido, A. (2004). Drosophila genetic variants that change cell size and rate of proliferation affect cell communication and hence patterning. Mech. Dev. 121(4): 351-64. 15110045

Rommel, C., et al. (1999). Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt. Science 286: 1738-1741. PubMed Citation: 10576741

Rossi, F. and Gonzalez, C. (2012). Synergism between altered cortical polarity and the PI3K/TOR pathway in the suppression of tumour growth. EMBO Rep 13: 157-162. PubMed ID: 22173033

Rubio, I. and Wetzker, R. (2000). A permissive function of phosphoinositide 3-kinase in Ras activation mediated by inhibition of GTPase-activating proteins Curr. Biol. 10: 1225-1228. 11050394

Rusten, T. E., et al. (2004). Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Dev. Cell 7: 179-192. 15296715

Ryu, B. R., et al. (1999). Phosphatidylinositol 3-kinase-mediated regulation of neuronal apoptosis and necrosis by insulin and IGF-I. J. Neurobiol. 39(4): 536-46

Sakaue, H., et al. (1998). Posttranscriptional control of adipocyte differentiation through activation of phosphoinositide 3-kinase. J. Biol. Chem. 273(44): 28945-52

Sander, E. E., et al. (1998). Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J. Cell Biol. 143(5): 1385-98

Sasson, E. E. and Stern, M. J. (2004), FGF and PI3 kinase signaling pathways antagonistically modulate sex muscle differentiation in C. elegans. Development 131: 5381-5392. 15469970

Saucedo, L. J., et al. (2003). Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5(6):566-71. 12766776

Schmelzle, T. and Hall, M. N. (2000). TOR, a central controller of cell growth. Cell 103: 253-262. 11057898

Schmid, A., Hallermann, S., Kittel, R. J., Khorramshahi, O., Frolich, A. M., Quentin, C., Rasse, T. M., Mertel, S., Heckmann, M. and Sigrist, S. J. (2008). Activity-dependent site-specific changes of glutamate receptor composition in vivo. Nat Neurosci 11: 659-666. PubMed ID: 18469810

Shi, P., Lai, R., Lin, Q., Iqbal, A. S., Young, L. C., Kwak, L. W., Ford, R. J. and Amin, H. M. (2009). IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells. Blood 114: 360-370. PubMed ID: 19423729

Shi, S. H., Jan, L. Y., and Jan, Y. N. (2003). Hippocampal neuronal polarity specified by spatially localized mPar3/mPar6 and PI 3-kinase activity. Cell 112: 63-75. 12526794

Shioi, T., et al. (2000). The conserved phosphoinositide 3-kinase pathway determines heart size in mice. EMBO J. 19: 2537-2548.

Shtivelman, E., Sussman, J. and Stokoe, D. (2002). A role for PI 3-Kinase and PKB activity in the G2/M phase of the cell cycle. Curr. Biol. 12: 919-924. 12062056

Sizemore, N., Leung, S. and Stark, G. R. (1999). Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-kappaB p65/RelA subunit. Mol. Cell. Biol. 19(7): 4798-805. PubMed ID: 10373529

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: 508-512. PubMed ID: 21346761

Sprinzak, D., et al. (2010). Cis-interactions between Notch and Delta generate mutually exclusive signalling states. Nature 465: 86-90. PubMed Citation: 20418862

Staveley, B. E., et al. (1998). Genetic analysis of protein kinase B (AKT) in Drosophila. Curr. Biol. 8(10): 599-602

Stolovich, M., Tang, H., Hornstein, E., Levy, G., Cohen, R., Bae, S. S., Birnbaum, M. J. and Meyuhas, O. (2002). Transduction of growth or mitogenic signals into translational activation of TOP mRNAs is fully reliant on the phosphatidylinositol 3-kinase-mediated pathway but requires neither S6K1 nor rpS6 phosphorylation. Mol. Cell. Biol. 22: 8101-8113. 12417714

Sun, H., et al. (2000). Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak. Curr. Biol. 10: 281-284.

Suzuki, H., et al. (1999). Xid-like immunodeficiency in mice with disruption of the p85alpha subunit of phosphoinositide 3-kinase. Science 283(5400): 390-2

Takahashi-Tezuka, M., et al. (1998). Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase. Mol. Cell. Biol. 18(7): 4109-17

Takuwa, N., Fukui, Y. and Takuwa, Y. (1999). Cyclin D1 expression mediated by phosphatidylinositol 3-kinase through mTOR-p70(S6K)-independent signaling in growth factor-stimulated NIH 3T3 fibroblasts. Mol. Cell. Biol. 19(2): 1346-58

Tamemoto, H. et al. (1994). Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature 372(6502): 182-6

Tang, H., Hornstein, E., Stolovich, M., Levy, G., Livingstone, M., Templeton, D., Avruch, J. and Meyuhas, O. (2001). Amino acid-induced translation of TOP mRNAs is fully dependent on PI3-kinase-mediated signaling, is partially inhibited by rapamycin, and is independent of S6K1 and rpS6 phosphorylation. Mol. Cell. Biol. 21: 8671-8683. 11713299

Tang, M.-J., et al. (2002). Ureteric bud outgrowth in response to RET activation is mediated by Phosphatidylinositol 3-kinase. Dev. Biol. 243: 128-136. 11846482

Tengholm, A. and Meyer, T. (2002). A PI3-kinase signaling code for Insulin-triggered insertion of glucose transporters into the plasma membrane. Curr. Biol. 12: 1871-1876. 12419189

Thakker, G. D.., et al. (1999). The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. J. Biol. Chem. 274(15): 10002-7

Valverde, A. M., et al. (1998). Insulin receptor substrate (IRS) proteins IRS-1 and IRS-2 differential signaling in the insulin/insulin-like growth factor-I pathways in fetal brown adipocytes. Mol. Endocrinol. 12(5): 688-97

van Weeren, P. C., et al. (1998). Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB. J. Biol. Chem. 273(21): 13150-6

Varnai, P., Rother, K. I. and Balla T. (1999). Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells. J. Biol. Chem. 274(16): 10983-9

Verdu, J., Buratovich, M. A., Wilder, E. L., and Birnbaum, M. J. (1999). Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB. Nat. Cell Biol. 1: 500-506. 10587646

Vachias, C., Fritsch, C., Pouchin, P., Bardot, O. and Mirouse, V. (2014). Tight coordination of growth and differentiation between germline and soma provides robustness for Drosophila egg development. Cell Rep 9: 531-541. ID: PubMed

Wang, X., Adam, J. C. and Montell, D. (2007). Spatially localized Kuzbanian required for specific activation of Notch during border cell migration. Dev. Biol. 301: 532-540. PubMed Citation: 17010965

Weiner, O. D., et al. (2002). A PtdInsP3- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity, Nat. Cell Biol. 4: 509-513. Medline abstract: 12080346

Weinkove, D., et al. (1997). p60 is an adaptor for the Drosophila phosphoinositide 3-kinase, Dp110. J. Biol. Chem. 272(23): 14606-10

Weinkove, D., et al. (1999). Regulation of imaginal disc cell size, cell number and organ size by Drosophila class IA phosphoinositide 3-kinase and its adaptor Curr. Biol. 9: 1019-1029

Willecke, M., Toggweiler, J. and Basler, K. (2011). Loss of PI3K blocks cell-cycle progression in a Drosophila tumor model. Oncogene 30(39): 4067-74. PubMed Citation: 21516128

Williams, E. J. and Doherty, P. (1999). Evidence for and against a pivotal role of PI 3-kinase in a neuronal cell survival pathway. Mol. Cell. Neurosci. 13(4): 272-80

Williams, K. D., Busto, M., Suster, M. L., So, A. K., Ben-Shahar, Y., Leevers, S. J. and Sokolowski, M. B. (2006). Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase. Proc. Natl. Acad. Sci. 103(43): 15911-5. PubMed citation: 17043223

Williams, M. R., et al. (2000). The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Current Biol. 10 : 439-448

Wittwer, F., et al. (2005). Susi, a negative regulator of Drosophila PI3-kinase. Dev Cell 8(6): 817-27. 15935772

Wong, C. C., et al. (2014). Inactivating CUX1 mutations promote tumorigenesis. Nat Genet 46: 33-38. PubMed ID: 24316979

Wood, W., Faria, C. and Jacinto, A. (2006). Distinct mechanisms regulate hemocyte chemotaxis during development and wound healing in Drosophila melanogaster. J Cell Biol. 173(3): 405-16 . PubMed citation; Online text

Yamada, M., et al. (1999). Brain-derived neurotrophic factor stimulates interactions of Shp2 with phosphatidylinositol 3-kinase and Grb2 in cultured cerebral cortical neurons. J. Neurochem. 73(1): 41-9. PubMed Citation: 10386953

Yan, J., et al. (1998). Ras isoforms vary in their ability to activate Raf-1 and phosphoinositide 3-kinase. J. Biol. Chem. 273(37): 24052-6. PubMed Citation: 9727023

Yang, H. and Raizada, M. K. (1999). Role of phosphatidylinositol 3-kinase in angiotensin II regulation of norepinephrine neuromodulation in brain neurons of the spontaneously hypertensive rat. J. Neurosci. 19(7): 2413-23. PubMed Citation: 10087056

Ye, K., et al. (2000). PIKE: A nuclear GTPase that enhances PI3kinase activity and is regulated by protein 4.1N. Cell 103: 919-930. PubMed Citation: 11136977

Xi, X., Tatei, K., Kihara, Y. and Izumi, T. (2014). Expression pattern of class I phosphoinositide 3-kinase and distribution of its product, phosphatidylinositol-3,4,5-trisphosphate, during Drosophila embryogenesis. Gene Expr Patterns. PubMed ID: 24928809

Xu, Z., et al. (2003). Maturation-associated increase in IP3 receptor type 1: role in conferring increased IP3 sensitivity and Ca2+ oscillatory behavior in mouse eggs. Dev. Bio. 254: 163-171. 12591238

Yenush, L., et al. (1996). The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis. Mol. Cell. Biol. 16(5): 2509-17

Yenush, L., and White, M.F. (1997). The IRS-signaling system during insulin and cytokine action. Bioessays 19: 491-500

Yoo, S. K., et al. (2010). Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev. Cell 18(2): 226-36. PubMed Citation: 20159593

Yoshii, A. and Constantine-Paton, M. (2007). BDNF induces transport of PSD-95 to dendrites through PI3K-AKT signaling after NMDA receptor activation. Nat. Neurosci. 10(6): 702-11. PubMed citation: 17515902

Yu, J., et al. (1998). Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. Mol. Cell. Biol. 18(3): 1379-87. PubMed Citation: 9488453

Yuan, T. L., Wulf, G., Burga, L. and Cantley, L. C. (2011). Cell-to-cell variability in PI3K protein level regulates PI3K-AKT pathway activity in cell populations. Curr. Biol. 21(3): 173-83. PubMed Citation: 21256021

Zhang, H., et al. (2000). Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 14: 2712-2724. 11069888

Zhang, S. Q., et al. (2002). Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2. Mol. Cell. Biol. 22(12): 4062-72. 12024020

Zhang, W., Thompson, B. J., Hietakangas, V. and Cohen S. M. (2011). MAPK/ERK signaling regulates insulin sensitivity to control glucose metabolism in Drosophila. PLoS Genet. 7(12): e1002429. PubMed Citation: 22242005

Zhou, F.-Q., et al. (2004). NGF-induced axon growth is mediated by localized inactivation of GSK-3ß and functions of the microtubule plus end binding protein APC. Neuron 42: 897-912. 15207235

Zhu, C.C., Boone, J.Q., Jensen, P.A., Hanna, S., Podemski, L., Locke, J., Doe, C.Q., and O'Connor, M.B. (2008). Drosophila Activin- and the Activin-like product Dawdle function redundantly to regulate proliferation in the larval brain. Development 135: 513-521. PubMed Citation: 18171686

Zimmermann, S. and Moelling, K. (1999). Phosphorylation and regulation of Raf by Akt (Protein kinase B). Science 286: 1741-1744. PubMed Citation: 10576742

Phosphotidylinositol 3 kinase 92E: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 5 February 2015

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