See the embryonic expression pattern of Akt1 at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site
The temporal and spatial expression of the DRAC-PK/Akt1 transcripts during development has been examined. For Northern blot analysis two probes were used: the cDNA DRAC 7 and a probe from the 3`-untranslated region located between the first polyadenylation signal (nucleotides 4374-4379) and a polyadenylation signal at nucleotides 5013-5018 in the DRAC-PK genomic sequence. Whereas DRAC 7 recognizes all classes of transcripts, the 3'-end probe would detect only those that use the second polyadenylation signal. Northern analysis of total RNA isolated from staged collections of embryos and larvae and from adult female flies using DRAC 7 as a probe revealed the presence of two major transcripts of 2.7 and 3.9 kb. The 2.7-kb transcript is expressed at high levels in 0-3-h embryos and is also expressed in adult females, indicating that this is a maternally regulated mRNA. The 3.9-kb transcript is expressed throughout embryogenesis and larval stages and is also detected in early and late pupal stages. This transcript is also expressed in the adult female flies, indicating both maternal and zygotic regulation of gene expression. When the 3'-probe was used, only the larger transcript could be detected, suggesting that the 2.7-kb transcript is generated by use of the first polyadenylation signal. This indicates that the cDNA SDE-RAC 109 is derived from the 2.7-kb maternal transcript (Andjelkovic, 1995).
In situ hybridization to whole-mount embryos and dissected ovaries, using DRAC 7 as a probe, demonstrates that maternally provided DRAC-PK transcripts are synthesized in the nurse cells of the ovaries. During oocyte maturation there is no apparent localization of the mRNA. No variation in expression could be detected throughout embryogenesis. Before cellularization, after cellularization, and throughout gastrulation, germ band extension, and retraction, DRAC-PK/Akt1 transcripts remain uniformly distributed and are expressed at a high level (Andjelkovic, 1995).
Northern analysis demonstrates that the DRAC-PK gene expression is both maternally and zygotically regulated. Also Western blot analysis reveals differences in DRAC-PK protein forms and abundance between embryos and adult flies. The expression of the DRAC-PK gene protein products during the Drosophila life cycle was examined. The major form detected throughout development is DRAC-PK66. The highest level of expression (arbitrarily taken as 100%) is found in 0-3-h embryos and subsequently declines to 60% during late embryogenesis. A further decline in DRAC-PK66 levels is observed during larval development (20% in the third instar larvae), followed by a sharp increase in the early pupae (80%). The protein levels are 30% in adult flies. DRAC-PK85 follows the expression of DRAC-PK66, except in the three larval stages where it could not be detected. There is no difference in the expression of DRAC-PK66 and -85 between female and male flies. p120 is detected during embryogenesis, where its expression follows the pattern of DRAC-PK66 and -85. It is found to be significantly higher in early than in late pupae, which is not the case with DRAC-PK66 and -85. These results suggest the involvement of DRAC-PK and the p120 protein in embryogenesis, as well as postembryonically. The spatial expression of the DRAC-PK proteins during embryogenesis was analyzed using the affinity-purified antirecombinant antibody No. 36. Proteins are uniformly distributed in all embryonic stages, which is in good correlation with the transcript localization. Also, during the syncytial blastoderm stage, specific staining is detected in the cytoplasm surrounding dividing nuclei and is always excluded from the chromatin (Andjelkovic, 1995).
The insulin/insulin-like growth factor signalling (IIS) cascade performs a broad range of evolutionarily conserved functions, including the regulation of growth, developmental timing and lifespan, and the control of sugar, protein and lipid metabolism. Recently, these functions have been genetically dissected in Drosophila, revealing a crucial role for cell-surface activation of the downstream effector kinase Akt in many of these processes. However, the mechanisms regulating lipid metabolism and the storage of lipid during development are less well characterized. The nutrient-storing nurse cells of the fly ovary were used to study the cellular effects of intracellular IIS components on lipid accumulation. These cells normally store lipid in a perinuclear pool of small neutral triglyceride-containing droplets. Loss of the IIS signalling antagonist PTEN, which stimulates cell growth in most developing tissues, produces a very different phenotype in nurse cells, inducing formation of highly enlarged lipid droplets. Furthermore, the accumulation of activated Akt in the cytoplasm is responsible for this phenotype and leads to a much higher expression of LSD2, the fly homologue of the vertebrate lipid-storage protein perilipin. This work therefore reveals a signalling mechanism by which the effect of insulin on lipid metabolism could be regulated independently of some of its other functions during development and adulthood. It is speculated that this mechanism could be important in explaining the well-established link between obesity and insulin resistance that is observed in Type 2 diabetes (Vereshchagina, 2006).
These data suggest that the effect of cytoplasmic P-Akt on lipid storage may be cell type-specific. Increasing IIS throughout the whole organism in viable Pten mutants surprisingly reduces total lipid content, whereas decreasing IIS elevates lipid levels both in flies and mice. It has not been possible to show biochemically whether triglycerides are increased rather than merely redistributed in Pten mutant ovaries, because only a small minority of the nurse cells is mutant. However, in some late-stage clones, in which cytoplasmic volumes are decreasing, lipid levels often appear elevated in mutant cells. Insulin-stimulated increases in lipid-droplet number and size have been observed in mammals, and, interestingly, when tumours are induced in mouse liver by raising IIS, lipid-storage mechanisms are also activated. It is proposed that these cell type-specific responses are due to selective accumulation of an IIS-modulated, cytoplasmic activated Akt pool that must, to some extent, be controlled independently of cell-surface P-Akt. Indeed, a Drosophila phosphatase has been identified that regulates levels of cytoplasmic P-Akt in nurse cells, and it has been shown that mutations in this gene produce a very similar enlarged lipid-droplet phenotype, confirming this hypothesis (Vereshchagina, 2006).
How could elevated cytoplasmic P-Akt induce such a dramatic lipid-droplet phenotype in nurse cells? In mammals, Akt can promote the transcription of genes involved in lipid biosynthesis and storage pathways. The data indicate LSD2/perilipin is one of these targets. IIS also post-translationally upregulates the activity of mammalian perilipin. Interestingly, ovaries mutant for Lsd2 show altered lipid accumulation, but droplets are still formed. Therefore, LSD2 is almost certainly one, but not the only, target for IIS in the control of lipid-droplet accumulation in nurse cells. In this context, it is interesting to note that, in addition to its proposed role in coating lipid droplets, LSD2 has recently been shown to regulate microtubule-dependent trafficking of these organelles. Since cytoplasmic P-Akt could still be associated with intracellular membranes or the droplet surface, it may be well positioned to modulate this transport process (Vereshchagina, 2006).
Obesity is a well-established predisposing factor in the acquisition of cellular insulin resistance and Type 2 diabetes. Increased levels of circulating free fatty acids (FFAs) associated with obesity appear to be important in this link. However, it is unclear whether other mechanisms are also involved or how reduced insulin sensitivity ultimately impacts on lipid storage. Molecules downstream of Akt are known to regulate cell-surface IIS through at least two negative-feedback loops involving downstream S6 kinase and the transcription factor FOXO. This work therefore raises the possibility that any predisposition towards increased cytoplasmic P-Akt could specifically promote lipid storage and also selectively suppress insulin-dependent events at the cell surface. It will be interesting to investigate further the molecules involved in controlling this P-Akt pool and whether the feedback mechanisms have any role to play in linking obesity and insulin resistance in Type 2 diabetes (Vereshchagina, 2006).
Drosophila peripheral nerves, structured similarly to their mammalian counterparts, comprise a layer of motor and sensory axons wrapped by an inner peripheral glia (analogous to the mammalian Schwann cell) and an outer perineurial glia (analogous to the mammalian perineurium). Growth and proliferation within mammalian peripheral nerves are increased by Ras pathway activation: loss-of-function mutations in Nf1, which encodes the Ras inhibitor neurofibromin, cause the human genetic disorder neurofibromatosis, which is characterized by formation of neurofibromas (tumors of peripheral nerves). However, the signaling pathways that control nerve growth downstream of Ras remain incompletely characterized. This study shows that expression specifically within the Drosophila peripheral glia of the constitutively active RasV12 increases perineurial glial thickness. Using chromosomal loss-of-function mutations and transgenes encoding dominant-negative and constitutively active proteins, it was shown that this nonautonomous effect of RasV12 is mediated by the Ras effector phosphatidylinositol 3-kinase (PI3K) and its downstream kinase Akt. The nonautonomous, growth-promoting effects of activated PI3K are suppressed by coexpression within the peripheral glia of FOXO, a transcription factor inhibited by Akt-dependent phosphorylation. It is suggested that Ras-PI3K-Akt activity in the peripheral glia promotes growth of the perineurial glia by inhibiting FOXO. In mammalian peripheral nerves, the Schwann cell releases several growth factors that affect the proliferative properties of neighbors. Some of these factors are oversecreted in Nf1 mutants. These results raise the possibility that neurofibroma formation in individuals with neurofibromatosis might result in part from a Ras-PI3K-Akt-dependent inhibition of FOXO within Schwann cells (Lavery, 2007).
Activating Ras specifically within the peripheral glia was sufficient to increase growth of the perineurial glia. In addition, activating the Ras effector PI3K, but not Raf or Ral, within the peripheral glia was sufficient to increase perineurial glial growth, and inhibiting PI3K activity in the peripheral glia, but not perineurial glia, suppressed the growth-promoting effects of activated Ras. It was also found that activity within the peripheral glia of the PI3K-activated kinase Akt was both necessary and sufficient to mediate the growth-promoting effects of PI3K. Finally, it was found that overexpression within the peripheral glia of FOXO, the forkhead-box transcription factor that is phosphorylated and inactivated by Akt-dependent phosphorylation, was sufficient to suppress the growth-promoting effects of PI3K. Together, these results suggest that Ras activity in the peripheral glia activates nonautonomous growth via the PI3K and Akt-dependent inhibition of FOXO. This observation is consistent with the previous observations that Nf1– mouse Schwann cells oversecrete growth factor(s) that cause increased recruitment of mast cells into the peripheral nerve and is consistent in part with the observation that the proliferation defects of Nf1– mutant mouse or human cells requires hyperactivation of Tor in a PI3K- and Akt-dependent manner (Lavery, 2007).
Perineurial glial growth in Drosophila peripheral nerves is regulated by several genes. These genes include Nf1, which is the Drosophila ortholog of human Nf1, push, which is thought to encode an E3 ubiquitin ligase and two genes implicated in neurotransmitter signaling: amnesiac, which is thought to encode a neuropeptide similar to vertebrate pituitary adenylate cyclase-activating polypeptide, and inebriated (ine), which encodes a member of the Na+/Cl–-dependent neurotransmitter transporter family. Some of these genes might regulate perineurial glial growth via the activity of Ras or PI3K within peripheral glia. For example, mutations in push, but not ine, enhance the perineurial glial growth phenotype of RasV12 expressed in peripheral glia. These observations are consistent with the possibility that the activity of ine regulates Ras-GTP levels within peripheral glia. In contrast, push might regulate PI3K in a Ras-independent manner or act in the perineurial glia to regulate sensitivity to peripheral glial growth factors. Additional experiments will be required to distinguish between these possibilities (Lavery, 2007).
There are several lines of evidence from mice and humans suggesting that cell nonautonomous growth regulation, as a consequence of intercellular signaling, underlies neurofibroma formation. First, although neurofibromas arise in individuals heterozygous for Nf1– after loss of Nf1+ from cell(s) within peripheral nerves, neurofibromas are heterogeneous and contain cells that are not clonally related, such as Schwann cells, perineurial cells, and fibroblasts. These observations suggest that neurofibromas arise when a core of Nf1– cells cause overproliferation of their heterozygous neighbors via nonautonomous means. Second, neurofibroma formation in mice and humans requires a homozygous Nf1 mutant genotype in Schwann cells but not other cells within the tumor. Third, Ras-GTP levels in Schwann cells from the mouse Nf1 knock-out mutant are uniformly elevated. In contrast, only a subset of Schwann cells from human neurofibromas exhibit elevated Ras- GTP levels; the possibility has been raised that this subset, but not other Schwann cells from the tumor, is homozygous for Nf1–. In this view, these Nf1– cells recruited neighboring Schwann cells that were heterozygous for Nf1– into the tumor by nonautonomous means, such as by the excessive release of one or more growth factors. Fourth, it has been demonstrated that Nf1– Schwann cells oversecrete the ligand for the c-Kit receptor. This oversecretion increased migration of mast cells into peripheral nerves and might be an essential step in neurofibroma formation. These Schwann cells also oversecrete additional factors whose physiological role remains unclear. The molecular mechanisms by which neurofibromin regulates the synthesis or release of these molecules remain incompletely understood. The current observations that Ras activity in the peripheral glia promotes growth nonautonomously via the PI3K- and Akt-dependent inhibition of FOXO might provide insights into the mechanisms by which peripheral nerve growth is regulated nonautonomously by the mammalian Schwann cell (Lavery, 2007).
By hyperactivating Ras, Nf1 mutations could in principle cause tumors via any of several Ras effector pathways. In addition, the diverse types of tumors observed in individuals with neurofibromatosis could result from hyperactivation of distinct Ras effector pathways. The Raf pathway has been viewed as a more relevant effector pathway than the PI3K pathway, mostly because the importance of Ras in the activation of PI3K under physiological conditions remains controversial. In particular, although it is clear that the oncogenic RasV12 mutant is sufficient to activate PI3K, it has sometimes been difficult to demonstrate that wild-type Ras is necessary for PI3K activation. Presumably, this difficulty reflects the fact that PI3K can be activated by Ras-independent as well as Ras-dependent mechanisms, such as direct activation by activated receptor tyrosine kinases or by PIKE-L (phosphatidylinositol kinase enhancer). However, more recently, it has been demonstrated that PI3K and Akt are hyperactivated in several Nf1 mutant cell types and that this hyperactivation is Ras dependent. Furthermore, PI3K activation plays an essential functional role in Nf1–-mediated growth defects, as is demonstrated by the observation that PI3K- and Akt-dependent Tor activation is necessary for the proliferation defects of Nf1 mutants to occur: application of rapamycin, a Tor inhibitor, attenuates the ability of Nf1 mutant cells to proliferate. These observations demonstrate that PI3K and Akt play key roles in at least some aspects of Nf1–-induced tumor growth (Lavery, 2007).
The results are consistent with these observations. By comparing the effects on perineurial glial growth of peripheral–glial expression of activated Raf, PI3K, or Ral, it was possible to demonstrate that activation of PI3K, not Raf or Ral, is sufficient to promote perineurial glial growth and that PI3K activity in the peripheral glia is necessary to observe the nonautonomous effect of activated Ras on perineurial glial growth. It was similarly shown that Akt activity os necessary and sufficient to mediate the growth-promoting effects of PI3K. However, previous studies have observed that Tor activation is critical for the PI3K- and Akt-dependent growth regulation of Nf1 mutant cells, this study observed a critical role for the PI3K- and Akt-dependent inhibition of the transcription factor FOXO. It is possible that the phenotype observed in previous studies reflects the well characterized ability of PI3K–Tor to activate growth cell autonomously, whereas the phenotype reported in this study reflects nonautonomous growth regulation. In this view, PI3K and Akt regulate autonomous and nonautonomous growth via the Tor and FOXO pathways, respectively (Lavery, 2007).
FOXO presumably inhibits the growth-promoting effects of PI3K and Akt by transcriptional regulation of target genes. Candidate FOXO target genes include those encoding the molecules oversecreted by Nf1– Schwann cells, whereas other targets might be represented in the distinct transcript profiles exhibited by Nf1– Schwann cells or malignant peripheral nerve sheath tumors compared with wild-type Schwann cells. For example, Schwann cells from neurofibromas, but not normal Schwann cells, express the epidermal growth factor (EGF) receptor. Other potential targets include genes encoding growth factors, although ectopic expression within the peripheral glia of two candidate genes, Hedgehog and the EGF ligands spitz and gurken, failed to induce perineurial glial growth. Additional experiments will be required to identify the FOXO targets that regulate nonautonomous growth in peripheral nerves (Lavery, 2007).
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).
The decision between survival and death is an important aspect of cellular regulation during development and malignancy. Central to this regulation is the process of apoptosis, which is conserved in multicellular organisms. A variety of signaling cascades have been implicated in the modulation of apoptosis, including the phosphatidylinositol (Pl) 3-kinase pathway. Activation of Pl 3-kinase is protective, and inhibition of this lipid kinase enhances cell death under several conditions, including deregulated expression of c-Myc, neurotrophin withdrawal and anoikis. Recently, the protective effects of Pl 3-kinase have been linked to its activation of the pleckstrin homology (PH)-domain-containing protein kinase B (PKB or AKT). PKB/AKT was identified from an oncogene, v-akt, found in a rodent T-cell lymphoma. To initiate a genetic analysis of PKB, a Drosophila PKB/AKT mutant (termed Dakt1) was isolated and characterized. It exhibits ectopic apoptosis during embryogenesis as judged by induction of membrane blebbing, DNA fragmentation and macrophage infiltration. These data implicate Dakt1 as a cell survival gene in Drosophila, consistent with cell protection studies in mammals (Staveley, 1998).
A single amino-acid change, F327I, was found to be a kinase dead mutant form of Akt1. This phenylalanine (F) residue is a core residue in subdomain VII of the kinase catalytic domain, forming the 'DFG' motif which is highly conserved among protein kinases including mammalian PKB proteins. Since Dakt1 is a maternally expressed gene, the maternal contribution of Dakt1 was tested using germline clone (GLC) analysis. GLC q females, containing clones of cells bearing the F327I mutation, produce embryos that lack various portions of larval cuticle by the end of embryogenesis. The range of phenotypes depends on the level of zygotic Dakt1 activity in the embryo. Without any zygotic expression of Dakt1, q GLC embryos produce only a few scraps of cuticle. When q GLC embryos express some zygotic Dakt1, some ventral cuticle is produced. This phenotype is extensively suppressed by expression of Dakt1 using a heat-shock inducible hs-Dakt1 transgene. Since mammalian PKB/AKT has been implicated in anti-apoptotic activity, a test was performed of Dakt1 GLC embryos for evidence of apoptosis. Acridine orange (AO) staining has been shown to be a good indicator of apoptosis (and not necrosis) in Drosophila, detecting cellular events such as membrane blebbing. AO staining of Dakt1 GLC embryos shows extensive apoptosis compared to wild-type embryos. Confirmation of an effect on apoptosis was performed using a TUNEL assay in Dakt1 embryos to detect the incidence of DNA fragmentation. Dakt1 embryos shows extensive DNA breakage as assayed by TUNEL in situ. Incidence of TUNEL signal in Dakt1 GLC embryos precedes the initiation of the signal in wild-type embryos. TUNEL signal accumulates during development of Dakt1 embryos to engulf the majority of the embryo. During apoptosis, macrophages converge to engulf cellular fragments by phagocytosis. Antibody to Croquemort (Crq), the Drosophila homolog of CD36, was used to detect macrophages in embryos. Dakt1 embryos exhibit a significant increase in Crq expression, as compared to wild-type embryos. This expression is at the cell surface and focuses on the apoptotic cells. It appears, therefore, that loss of Dakt1 activity results in premature and ectopic apoptosis with the characteristics of membrane blebbing, DNA fragmentation and macrophage-mediated endocytosis (Staveley, 1998).
The deficiency of the reaper (rpr), grim and hid genes [Df(3L)H99] blocks apoptosis in Drosophila. Overexpression of any of these three genes results in ectopic apoptosis in embryos. It was therefore of interest to see whether Dakt1 mutants result in apoptosis through the mis-expression of rpr, grim or hid. This was found not to be the case, since Dakt1 mutant embryos do not show overexpression of these genes. Loss of rpr, grim and hid in H99 do not suppress the phenotype of Dakt1 GLC embryos. These results suggest that Dakt1 and H99 modulate apoptosis via distinct mechanisms. To test the involvement of caspases in Dakt1-mediated apoptosis, the baculoviral caspase-inhibitory protein p35 was expressed in Dakt1 embryos. Ectopic p35 has been shown to block caspase activity and suppress apoptosis in Dakt1 embryos, and hs-p35 effectively blocks apoptosis in Dakt1 embryos, demonstrating the requirement for caspase activity in this process (Staveley, 1998).
These epistasis tests suggest that Dakt1 does not function upstream of the rpr, grim and hid gene functions in the embryo. It is possible, though, that Dakt1 might be regulated by the rpr, grim and hid genes (at the H99 locus) and in fact act downstream of these genes. This presents two possibilities: (1) Dakt1 and the H99 locus represent independent pathways; (2) the H99 locus might repress Dakt1 function. This study thus provides the first genetic evidence implicating PKB as an anti-apoptotic factor (Staveley, 1998).
To determine whether Akt1 transduces growth-related signals, Akt1-deficient somatic clones were generated in the developing eye by mitotic recombination. Adult eyes exhibit a reduction in size in Akt1-deficient rhabdomeres, which, in some instances, co-exist with normal-sized heterozygous cells in the same ommatidium. Akt1 mutant clones are rare and small and are obtained only after heat-shock during the third instar larval stage. These observations indicate that the lack of Akt1 clones in the adult retina following induction at early larval stages may have resulted from cell competition, by which the Akt1-deficient cells would be eliminated and replaced by the surrounding wild-type sister cells. The phenotype of the Akt1-deficient rhabdomeres may have resulted from perturbations of cell growth or proliferation. The smaller size of these rhabdomeres shows that Akt1 is essential for normal cell growth, but dispensable for cell-fate determination. Moreover, the co-existence of Akt1 mutant rhabdomeres with wild-type twin-spot rhabdomeres in the same ommatidium suggests a cell-autonomous control of cell growth by Akt1 (Verdu, 1999).
The small size of the clones of Akt1-deficient cells could be the result of an impairment in the proliferation, survival, or both, of homozygous null cells. To evaluate more specifically the effects of Akt1 on proliferation or growth in vivo, upstream activation sequence (UAS)-Akt1 lines were generated with which to investigate the effects of altering the amount of Akt1 during Drosophila eye and wing imaginal disc development. The gmrGAL4 transgene targets expression of Akt1 to cells posterior to the morphogenetic furrow, producing flies exhibiting enlarged and bulging eyes with a mild disruption of the regular, external lattice. Similar, but less pronounced, effects are observed with a sevGAL4 transgene. Quantitative analysis reveals that the Akt1-induced increase in the size of the eye is caused by an increase in the size but not in the number of ommatidia. To determine the extent of this phenotype, tangential sections of these eyes were examined. In spite of the rough appearance of the adult compound eye, Akt1 expression does not affect the normal process of photoreceptor cell-fate determination in these larger ommatidia (Verdu, 1999).
Akt plays a central part in promoting the survival of a wide range of cell types in mammalian systems and in Drosophila embryos. However, overexpression of Akt1 does not alter the normal rate of apoptosis in the eye, as shown by equivalent acridine orange staining in control and gmrGAL4/UAS-Akt1 eye imaginal discs. Hence, overexpression of Akt1 affects neither the normal processes of cell-fate determination nor apoptosis in the developing retina (Verdu, 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).
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).
A mutation has been isolated in the Drosophila homolog of TSC1 (Tsc1). Cells mutant for Tsc1 are dramatically increased in size yet differentiate normally. Organ size is also increased in tissues that contain a majority of mutant cells. Clones of Tsc1 mutant cells in the imaginal discs undergo additional divisions but retain normal ploidy. Flow cytometry analysis indicates that the increase in cell size is not due to endoreplication. Tsc1 protein is shown to bind to Drosophila Tsc2 in vitro. Overexpression of Tsc1 or Tsc2 alone in the wing and eye has no effect, but co-overexpression leads to a decrease in cell size, cell number, and organ size. Genetic epistasis data are consistent with a model that Tsc1 and Tsc2 function together in the insulin signaling pathway (Potter, 2001).
Recent work has demonstrated that the insulin signaling pathway plays an important role in the regulation of cell size, cell number, and organ size. Mutations of Drosophila PTEN (dPTEN), which functions as a negative regulator of insulin signaling, result in phenotypes that resemble the effects of Tsc1 and Tsc2 mutations. Therefore, genetic epistasis experiments were performed to test whether Tsc1 or Tsc2 might also function to negatively regulate insulin signaling. Overexpression of Drosophila insulin receptor (dinr) using the eyGAL4 driver leads to lethality at 25°C and a dramatic increase in ommatidia number in escapers at room temperature. Co-overexpression of Tsc1 and Tsc2 (but not either Tsc1 or Tsc2 alone) rescues both the lethality and the extra ommatidia phenotype caused by dinr overexpression. Furthermore, overexpression of dinr using the pGMR-GAL4 driver leads to an increase in ommatidium size, which is also suppressed by co-overexpression of Tsc1 and Tsc2. Clones of dinr mutant ommatidia are smaller in size than wild-type. Ommatidia that are mutant for both dinr and Tsc1, however, exhibit the Tsc1 mutant phenotype of increased ommatidium size (Potter, 2001).
Overexpression of dPTEN using the pGMR-GAL4 driver leads to eyes with a decreased ommatidium size. However, overexpression of dPTEN is unable to suppress the clonal Tsc1 mutant phenotype. Similar to dinr, clones of dAkt mutant ommatidia are smaller in size. Ommatidia mutant for both dAkt and Tsc1 display the Tsc1 phenotype. Similarly, ommatidia that contained Tsc2 mutant clones in a dAkt mutant background exhibit the Tsc2 mutant phenotype. These results suggest that in the eye, Tsc1 and Tsc2 function genetically epistatic to (downstream of) dinr, dPTEN, and dAkt (Potter, 2001).
Genetic analyses suggest that the TSC genes act in a parallel pathway that converges on the insulin pathway downstream from Akt. The most convincing evidence for a functional link between the TSC genes and insulin signaling comes from the observation that heterozygosity of TSC1 or TSC2 is sufficient to rescue the lethality of loss-of-function InR mutants. This argues that the TSC genes are intimately linked to insulin signaling, rather than functioning in a totally independent cell-growth pathway. These results suggest that the TSC tumor suppressor genes are novel negative regulators of insulin signaling, and modulating the activities of the TSC genes might provide a potential way to correct insulin signaling defects in certain diseases such as diabetes and obesity (Gao, 2001).
Previous studies have shown that loss of inr or Akt leads to decreased cell size. To investigate the relationship between inr, Akt, and the TSC genes, TSC1;Akt and TSC1;inr double-mutant clones were studied. Cells homozygous for a strong allele of inr, or a null allele of Akt are smaller, and are rarely recovered in adult eye clones because of cell competition during development. However, TSC1;inror TSC1;Akt1 double-mutant cells showed a similar cell size increase as that observed in TSC1- cells. Furthermore, the competitive disadvantage of inr and Akt mutant cells is also rescued in the TSC1;inr or TSC1;Akt1 double-mutant clones, resulting in larger clones that contained more cells. This result suggests that TSC1 acts genetically downstream from Akt. This observation is compatible with either TSC1 acting molecularly downstream from Akt in the linear InR-PI3K-Akt pathway, or TSC1 acting in a parallel pathway that converges on the insulin pathway downstream from Akt (Gao, 2001).
The Drosophila gene chico encodes an insulin receptor substrate that functions in an insulin/insulin-like growth factor (IGF) signaling pathway. In the nematode C. elegans, insulin/IGF signaling regulates adult longevity. Mutation of chico extends fruit fly median life-span by up to 48% in homozygotes and 36% in heterozygotes. Extension of life-span is not a result of impaired oogenesis in chico females, nor is it consistently correlated with increased stress resistance. The dwarf phenotype of chico homozygotes was also unnecessary for extension of life-span. The role of insulin/IGF signaling in regulating animal aging is therefore evolutionarily conserved (Clancy, 2001).
In Drosophila, the insulin/IGF receptor INR, the insulin receptor substrate Chico, the phosphatidylinositol 3-kinase (PI3K) Dp110/p60, and the PI3K target protein kinase B (PKB, also known as DAkt1) form a signaling pathway that regulates growth and size. The effects on aging of hypomorphic mutations in Inr (equivalent to daf-2) and PKB, and null mutations in chico and the catalytic (Dp110, equivalent to age-1) and adapter (p60) PI3K subunits were examined. All mutants were tested as heterozygotes. chico1 and PKB3 homozygotes and InrGC25/InrE19 transheterozygotes, which form viable dwarf adults, were also examined. The remaining mutations were homozygous lethal (Clancy, 2001).
Most mutants tested had normal or significantly decreased life-span. For example, PKB3 homozygotes and InrGC25/InrE19 flies are short-lived. By contrast, chico1 extends life-span. Homozygous chico1 females exhibit an increase of median and maximum life-span of up to 48% and 41%, respectively. chico1 heterozygotes also exhibit increases in median life-span of up to 36% and 13% in females and males, respectively. Homozygous males, however, are slightly short-lived (Clancy, 2001).
Of the mutations tested, only chico1 increases life-span. This may be because the effect of reduced IIS on life-span depends on the degree to which signaling is reduced. Unlike the other null mutations in IIS genes tested, chico1 is not homozygous lethal, presumably because the INR receptor can signal to PI3K directly, as well as indirectly via Chico. Thus, chico1 mutants may be long-lived because of the relatively mild reduction in pathway activity that they bring about. Notably, severe IIS mutations in C. elegans can cause premature mortality in some adults, although the maximum life-span of populations is invariably increased. This is probably why InrGC25/InrE19 flies are short-lived: Demographic analysis indicates that a reduction in the age-specific mortality rate acceleration occurs, whose effect on survival is masked by an elevated rate of age-independent mortality. Furthermore, a different heteroallelic Drosophila Inr mutant to that tested here exhibits an 85% increase in female life-span. By contrast, in short-lived PKB3 populations, no reduction in mortality rate acceleration is seen. This raises the possibility that a second pathway downstream of chico might regulate aging in Drosophila. Interestingly, Chico contains potential binding sites for the Drk/Grb2 docking protein, consistent with signaling via Ras/mitogen-activated protein kinase (Clancy, 2001).
Analysis of Drosophila Foxo indicates that it is a critical PKB target, but that it mediates only one aspect of PKB function. Several lines of evidence support this model. (1) The effects of ectopic overexpression of Foxo and the human homolog hFOXO3a in the developing Drosophila eye are altered by Dp110 and PKB signaling as well as by nutrient levels. Under conditions of lowered insulin signaling, the phenotypes resulting from expression of foxo and hFOXO3a are dramatically enhanced. This situation was mimicked by expressing a PKB-insensitive phosphorylation mutant, suggesting that endogenous PKB signaling is required to mitigate the effects of ectopically expressed Foxo and hFOXO3a. (2) The physiological relevance of Foxo in PKB signaling is most vividly demonstrated by the observation that the larval lethality associated with the complete loss of PKB is rescued by foxo mutations to the extent that some flies develop to pharate adults. The lethality associated with loss of PKB function is therefore to a large extent due to the hyperactivation of Foxo. (3) Loss of Foxo function suppresses the effects of insulin-signaling mutations only partially; Foxo mediates a reduction in cell number but not in cell size in response to reduced insulin signaling (Jünger, 2003).
Genetic analysis of the control of body size in Drosophila has revealed two classes of mutations. Flies carrying mutations in chico or viable allelic combinations of Inr, Dp110, and PKB are reduced in body size by up to 50% owing to a reduction in both cell size and cell number. Conversely, flies mutant for S6K exhibit a more moderate reduction in body size, caused almost exclusively by a reduction in cell size. This suggests that the pathways controlling cell number and cell size bifurcate at or below PKB. Although foxo single mutants have no obvious size phenotype, loss of foxo substantially suppresses the cell-number reduction observed in insulin-signaling mutants. It appears that Foxo mediates the repression of proliferation in flies mutant for Inr, chico, Dp110, and PKB without being required for the reduction in cell size. Chico-Foxo double mutant flies even have slightly smaller cells than chico mutants, suggesting that removal of Foxo permits cell-cycle acceleration under conditions of impaired insulin signaling. The pathway controlling body size in response to insulin therefore bifurcates at the level of PKB: PKB controls cell number by inhibiting Foxo function and PKB controls cell size, at least under some conditions, by regulating S6K activity by phosphorylation of TSC2 (Jünger, 2003).
The signaling systems controlling cell size and cell number are tightly interconnected. Genetic and biochemical analyses have revealed five different links between the TSC-TOR-S6K pathway and the Inr-PKB-Foxo pathway. (1) Under conditions of unnaturally high insulin-signaling activity (that is, following the oncogenic activation of PKB) PKB phosphorylates and inactivates TSC2, resulting in increased activation of S6K. Under normal culture conditions this regulation does not seem critical, however, loss of dPKB function does not lower dS6K activity in larval extracts. (2) Under physiological conditions, PDK1 regulates PKB as well as S6K. (3) S6K itself downregulates dPKB activity in a negative feedback loop. (4) Under severe starvation conditions, nuclear Foxo presumably activates target genes that reduce cell proliferation. One of these target genes is 4E-BP, which encodes an inhibitor of translation initiation. When conditions improve, the insulin and TOR signaling pathways can stimulate translation by disrupting the 4E-BP/eIF4E complex via phosphorylation of 4E-BP, and in parallel by repressing FOXO-dependent 4E-BP expression. (5) Under even more severe starvation or stress conditions, full activation of Foxo upregulates expression of the insulin receptor itself, thus rendering the cell hypersensitive to low insulin levels. These multiple positive and negative interactions ensure a continuous fine adjustment of the growth rate to changing environmental conditions (Jünger, 2003).
Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosopohila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. scylla and charybdis (a. k. a. charybde), two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The genes are named after mythological monsters that lived in the Strait of Messina between Sicily and Italy, posing a threat to the passage of ships. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, either scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC1. Furthermore, scylla and charybdis are induced under hypoxic conditions and scylla is a target of Drosopohila HIF-1 (hypoxia-inducible factor-1: Similar) like its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing (Reiling, 2004).
Although loss of Scylla function does not produce a mutant phenotype on its own, whether it would alter the PKB/PDK1 overexpression eye phenotype was tested. Indeed, loss of Scylla function enhances the PKB/PDK1 overgrowth phenotype. Thus, Scylla is essential for attenuating the increased growth in response to hyperactivation of the Inr pathway. Furthermore, loss of Scylla partially suppresses the growth reduction associated with reduced PKB function as assessed by comparing weights of PKB3 single mutants to scy31 PKB3 double mutants. In contrast, complete loss of Scylla in a heteroallelic S6K combination does not rescue the S6K single mutant phenotype indicating that S6K is epistatic over scylla (Reiling, 2004).
Moreover, verexpression of scylla and charybdis not only suppresses the growth phenotype caused by over-activation of the Inr pathway in the eye but to a certain extent also rescues the lethality associated with the ubiquitous increase in Inr pathway activity due to either overexpression of PKB or loss of PTEN. scylla rescues the male-specific lethality caused by ubiquitous expression of PKB and organismal lethality associated with the partial but not complete loss of PTEN function. Similarly, PKB-associated male lethality is also rescued by charybdis overexpression. This indicates that scylla and charybdis have the capacity to act as potent negative regulators of insulin signaling downstream of PKB and PDK1 (Reiling, 2004).
Several lines of evidence suggest that Scylla and Charybdis act upstream of TSC and Rheb. Tsc1/2 mutant flies can be rescued to adulthood by reducing S6K signaling, and a mere reduction of one TOR copy in a Tsc1 mutant context results in a rescue to the pupal stage. Whether ubiquitous scylla overexpression could rescue the larval lethality of heteroallelic Tsc1/2 mutant combinations (Tsc12G3/Tsc1Q87X and Tsc256/Tsc2192) was examined using the da-Gal4 or Act5C-Gal4 drivers in combination with a UAS-scy transgene or EPscy at 18°C, 25°C, and 29°C. Ubiquitous overexpression of scylla/charybdis in a Tsc1/2 mutant background did in no case extend larval development beyond first/second instar, and these larvae died at the same time as Tsc1/2 mutants. Moreover, the big head phenotype of Tsc2192 (and Tsc256) induced by the eyflp/FRT system was not further enhanced in scyEP9.85 char180 Tsc2 triple-mutant heads. It has been shown that heads composed almost entirely of scylla charybdis double-mutant cells are enlarged. Conversely, GMRGal4-driven co-overexpression of Tsc1, Tsc2, and scylla or charybdis in the eye does not further reduce the small eye phenotype induced by coexpression of Tsc1 and Tsc2 on their own. The absence of an additive growth effect upon loss of Tsc2, scylla, and charybdis or overexpression of Tsc1/2 and scylla or charybdis suggests that they function in the same pathway. These results are consistent with the idea that Scylla and Charybdis act upstream of the TSC complex. This conclusion is further supported by the fact that neither a Rheb-dependent bulging eye phenotype nor organismal lethality could be suppressed by scylla/charybdis coexpression (Reiling, 2004).
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