InteractiveFly: GeneBrief

Insulin-like receptor : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - Insulin-like receptor

Synonyms -

Cytological map position -

Function - receptor

Keywords - insulin receptor pathway, growth, survival

Symbol - InR

FlyBase ID: FBgn0283499

Genetic map position -

Classification - protein tyrosine kinase

Cellular location - surface

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Lebreton, S., Trona, F., Borrero-Echeverry, F., Bilz, F., Grabe, V., Becher, P. G., Carlsson, M. A., Nassel, D. R., Hansson, B. S., Sachse, S. and Witzgall, P. (2015). Feeding regulates sex pheromone attraction and courtship in Drosophila females. Sci Rep 5: 13132. PubMed ID: 26255707
In Drosophila melanogaster, gender-specific behavioural responses to the male-produced sex pheromone cis-vaccenyl acetate (cVA) rely on sexually dimorphic, third-order neural circuits. This study shows that nutritional state in female flies modulates cVA perception in first-order olfactory neurons. Starvation increases, and feeding reduces attraction to food odour, in both sexes. Adding cVA to food odour, however, maintains attraction in fed females, while it has no effect in males. Upregulation of sensitivity and behavioural responsiveness to cVA in fed females is paralleled by a strong increase in receptivity to male courtship. Functional imaging of the antennal lobe (AL), the olfactory centre in the insect brain, shows that olfactory input to DA1 and VM2 glomeruli is also modulated by starvation. Knocking down insulin receptors in neurons converging onto the DA1 glomerulus suggests that insulin-signalling partly controls pheromone perception in the AL, and adjusts cVA attraction according to nutritional state and sexual receptivity in Drosophila females.
Yu, Y., Huang, R., Ye, J., Zhang, V., Wu, C., Cheng, G., Jia, J. and Wang, L. (2016). Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila. Elife [Epub ahead of print]. PubMed ID: 27612383
Starvation induces sustained increase in locomotion, which facilitates food localization and acquisition and hence composes an important aspect of food-seeking behavior. This study investigated how nutritional states modulate starvation-induced hyperactivity in adult Drosophila. The receptor of adipokinetic hormone (AKHR), the insect analog of glucagon, is required for starvation-induced hyperactivity. AKHR is expressed in a small group of octopaminergic neurons in the brain. Silencing AKHR+ neurons and blocking octopamine signaling in these neurons eliminates starvation-induced hyperactivity, whereas activation of these neurons accelerates the onset of hyperactivity upon starvation. Neither AKHR nor AKHR+ neurons are involved in increased food consumption upon starvation, suggesting that starvation-induced hyperactivity and food consumption are independently regulated. Single cell analysis of AKHR+ neurons identified the co-expression of Drosophila insulin-like receptor (dInR), which imposes suppressive effect on starvation-induced hyperactivity. Therefore, insulin and glucagon signaling exert opposite effects on starvation-induced hyperactivity via a common neural target in Drosophila.

Wei, Y., Gokhale, R. H., Sonnenschein, A., Montgomery, K. M., Ingersoll, A. and Arnosti, D. N. (2016). Complex cis-regulatory landscape of the insulin receptor gene underlies the broad expression of a central signaling regulator. Development 143: 3591-3603. PubMed ID: 27702787
Insulin signaling plays key roles in development, growth and metabolism through dynamic control of glucose uptake, global protein translation and transcriptional regulation. Altered levels of insulin signaling are known to play key roles in development and disease, yet the molecular basis of such differential signaling remains obscure. Expression of the insulin receptor (InR) gene itself appears to play an important role, but the nature of the molecular wiring controlling InR transcription has not been elucidated. This study characterized the regulatory elements driving Drosophila InR expression and found that the generally broad expression of this gene is belied by complex individual switch elements, the dynamic regulation of which reflects direct and indirect contributions of FOXO, EcR, Rbf and additional transcription factors through redundant elements dispersed throughout approximately 40 kb of non-coding regions. The control of InR transcription in response to nutritional and tissue-specific inputs represents an integration of multiple cis-regulatory elements, the structure and function of which may have been sculpted by evolutionary selection to provide a highly tailored set of signaling responses on developmental and tissue-specific levels.
Tanabe, K., Itoh, M. and Tonoki, A. (2017). Age-related changes in Insulin-like signaling lead to intermediate-term memory impairment in Drosophila. Cell Rep 18: 1598-1605. PubMed ID: 28199832
Insulin and insulin-growth-factor-like signaling (IIS) plays important roles in the regulation of development, growth, metabolic homeostasis, and aging, as well as in brain functions such as learning and memory. The temporal-spatial role of IIS in learning and memory and its effect on age-dependent memory impairment remain unclear. This study reports that intermediate-term memory (ITM), but not short-term memory (STM), in Drosophila aversive olfactory memory requires transient IIS during adulthood. The expression of Drosophila insulin-like peptide 3 (Dilp3) in insulin-producing cells and insulin receptor function in the fat body are essential for ITM. Although the expression of dilp3 decreases with aging, which is unique among dilp genes, the transient expression of dilp3 in aged flies enhances ITM. These findings indicate that ITM is systemically regulated by communication between insulin-producing cells and fat body and that age-dependent changes in IIS contribute to age-related memory impairment.

Lebreton, S., Carlsson, M. A. and Witzgall, P. (2017). Insulin signaling in the peripheral and central nervous system regulates female sexual receptivity during starvation in Drosophila. Front Physiol 8: 685. PubMed ID: 28943854
Many animals adjust their reproductive behavior according to nutritional state and food availability. Drosophila females for instance decrease their sexual receptivity following starvation. Insulin signaling, which regulates many aspects of insect physiology and behavior, also affects reproduction in females. This study shows that insulin signaling is involved in the starvation-induced reduction in female receptivity. More specifically, females mutant for the insulin-like peptide 5 (dilp5) were less affected by starvation compared to the other dilp mutants and wild-type flies. Knocking-down the insulin receptor, either in all fruitless-positive neurons or a subset of these neurons dedicated to the perception of a male aphrodisiac pheromone, decreased the effect of starvation on female receptivity. Disrupting insulin signaling in some parts of the brain, including the mushroom bodies even abolished the effect of starvation. In addition, fruitless-positive neurons in the dorso-lateral protocerebrum and in the mushroom bodies co-expressing the insulin receptor were identified. Together, these results suggest that the interaction of insulin peptides determines the tuning of female sexual behavior, either by acting on pheromone perception or directly in the central nervous system.
Orengo, D. J., Aguade, M. and Juan, E. (2017). Characterization of dFOXO binding sites upstream of the Insulin Receptor P2 promoter across the Drosophila phylogeny. PLoS One 12(12): e0188357. PubMed ID: 29200426
The insulin/TOR signal transduction pathway plays a critical role in determining such important traits as body and organ size, metabolic homeostasis and life span. Although this pathway is highly conserved across the animal kingdom, the affected traits can exhibit important differences even between closely related species. Evolutionary studies of regulatory regions require the reliable identification of transcription factor binding sites. This study has focused on the Insulin Receptor (InR) expression from its P2 promoter in the Drosophila genus, which in D. melanogaster is up-regulated by hypophosphorylated Drosophila FOXO (dFOXO). Transcription factor binding sites were finely characterized in vitro along the 1.3 kb region upstream of the InR P2 promoter in five Drosophila species. Moreover, the effect of mutations in the characterized dFOXO sites of D. melanogaster was characterized in transgenic flies. The number of experimentally established binding sites varies across the 1.3 kb region of any particular species, and their distribution also differs among species. In D. melanogaster, InR expression from P2 is differentially affected by dFOXO binding sites at the proximal and distal halves of the species 1.3 kb fragment. The observed uneven distribution of binding sites across this fragment might underlie their differential contribution to regulate InR transcription.
Luhur, A., Buddika, K., Ariyapala, I. S., Chen, S. and Sokol, N. S. (2017). Opposing post-transcriptional control of InR by FMRP and LIN-28 adjusts stem cell-based tissue growth. Cell Rep 21(10): 2671-2677. PubMed ID: 29212015
Although the intrinsic mechanisms that control whether stem cells divide symmetrically or asymmetrically underlie tissue growth and homeostasis, they remain poorly defined. This study reports that the RNA-binding protein fragile X mental retardation protein (FMRP) limits the symmetric division, and resulting expansion, of the stem cell population during adaptive intestinal growth in Drosophila. The elevated insulin sensitivity that FMRP-deficient progenitor cells display contributes to their accelerated expansion, which is suppressed by the depletion of insulin-signaling components. This FMRP activity is mediated solely via a second conserved RNA-binding protein, LIN-28, known to boost insulin signaling in stem cells. Via LIN-28, FMRP controls progenitor cell behavior by post-transcriptionally repressing the level of Insulin receptor (InR). This study identifies the stem cell-based mechanism by which FMRP controls tissue adaptation, and it raises the possibility that defective adaptive growth underlies the accelerated growth, gastrointestinal, and other symptoms that affect fragile X syndrome patients.

Each individual organ grows by controlling cell number and/or cell size to reach its final dimensions in relation to the size of the organism. This process is tightly regulated and modulated by environmental factors such as nutrient availability and temperature. How organ growth is coordinated within a single individual is still poorly understood. In mammals, hormones and growth factors are known to play a predominant role in controlling organismal growth by orchestrating cell growth, cell proliferation, and cell survival. Reducing the levels of growth hormone or its mediators, insulin like growth factor (IGF1) and the IGF1 receptor (IGF1R), strongly affects body and organ size. In contrast to the well-established role of the IGF1R in growth control, a corresponding role of the insulin receptor is less well understood (Brogiolo, 2001 and references therein).

Genetic studies in Drosophila have highlighted an evolutionarily conserved signaling pathway that plays an essential role in controlling body, organ, and cell size. This pathway involves the homolog of the insulin receptor substrates (Chico), Phosphotidylinositol 3 kinase (Dp110), Pten, Akt/PKB, and RPS6-p70-protein kinase (S6K). Mutations in any one of these components lead to a change in cell size and, with the exception of S6K, in cell number as well. Conversely, overexpression of Dp110 or Akt1 leads to an increased cell size without affecting cell numbers. Thus, it appears that stimulation of the PI(3)K/PKB pathway alone is not sufficient to promote cell growth and cell cycle progression (Brogiolo, 2001 and references therein).

It is not known, however, whether the effect of InR on growth is cell autonomous and whether activation of InR is sufficient to promote growth and cell division. Furthermore, the identity of a ligand(s) for InR has remained elusive. It is now clear, however, that there are seven insulin-like genes in Drosophila and that these are expressed in a highly tissue- and stage-specific pattern. InR regulates organ size by changing cell size and cell number in a cell-autonomous manner. An amino acid substitution at the corresponding position in the kinase domain of the human and Drosophila insulin receptors causes severe growth retardation. Overexpression of one of the insulin-like genes alters growth control in an InR-dependent manner. With the discovery of the ligands for InR, research on the insulin receptor pathway function enters a new phase of clarity and biological interest (Brogiolo, 2001 and references therein).

The Drosophila homolog of the insulin/IGF1 receptor, InR, is essential for normal development and is required for the formation of the epidermis and the central and peripheral nervous systems during embryogenesis (Fernandez, 1995). All described alleles of InR are recessive embryonic or early larval lethal. Only weak heteroallelic combinations of InR alleles were found to be viable and yield adults with a severe developmental delay, small body size, and female sterility (Fernandez, 1995, Chen, 1996 and Brogiolo, 2001).

Flies that are homozygous for a partial loss-of-function mutation in InR (InRE19) (Chen, 1996) show a phenotype similar to that previously described for weak heteroallelic combinations. The developmental time is extended from 10 to 20 days, and body size is severely but proportionally reduced. The mutant flies are approximately half the weight of their heterozygous siblings, and females are sterile. Furthermore, like chico mutant flies, InRE19 flies have an almost 2-fold increase in lipid content. The small body size is attributable to a reduction in cell size and cell number by 23% and 17%, respectively as revealed by measuring cell density in the wing. Similarly, the average number of ommatidia in the compound eye of mutant male flies is 378 +/- 8 compared to 683 +/- 8 in heterozygous control flies. No dominant size reduction was observed with various InR alleles (Brogiolo, 2001).

The reduced overall size could be due to InR acting in the humoral regulation of growth or to it functioning autonomously in a cell- and tissue-specific manner. To test whether InR affects body parts autonomously, InR function in the eye imaginal disc was selectively removed using the ey-FLP technique. The eye imaginal disc gives rise to the adult eye and the head capsule. Mosaic flies with heads largely homozygous for various InR alleles display a dramatic reduction in eye tissue and in the head capsule, whereas the other body parts are of wild-type size. Notably, the head size is dependent on the allele. This allowed alleles to be arranged according to their phenotypic strength. The strongest reduction in head size was observed with InR339, a putative null allele, followed by InR31, InR211, InRE19, and InR353. Thus, InR regulates head size autonomously (Brogiolo, 2001).

A comparison of homozygous mutant tissue with heterozygous tissue in tangential sections of mosaic eyes reveals an estimated reduction in ommatidial size of one third for InRE19 homozygous mutant tissue and of more than half for a candidate null allele. Importantly, this growth defect does not impede proper cell fate determination, given that the normal arrangement of the photoreceptor rhabdomeres is retained. Furthermore, the cell size reduction is cell autonomous, as can be seen at the border between homozygous mutant tissue and heterozygous tissue; within the same ommatidial unit, small homozygous cells coexist with normal-sized heterozygous cells. Although cells lacking InR function survive and differentiate normally, they have a growth disadvantage compared to heterozygous cells. When homozygous mutant cell clones are induced during early larval life and analyzed in the imaginal discs in the third instar, clone size is greatly reduced, compared to the wild-type sister clone. The phenotypes of InR mutant cells are strikingly similar to those of mutants in the Pi(3)K/PKB pathway. Therefore, it is likely that InR directly regulates cell growth at least in part through the Pi(3)K/PKB pathway (Brogiolo, 2001).

The structure of InR is similar to the mammalian insulin receptor (Inr) and the IGF1 receptor (IGF1R). It is a tetramer composed of two subunits containing the putative ligand binding domains and two transmembrane subunits containing the cytoplasmic tyrosine kinase domains. In contrast to human receptors, InR possesses extensions at the amino and carboxy termini. The C-terminal extension contains binding sites for downstream components similar to those found in insulin receptor substrates (IRS), and has been shown to be able to signal in the absence of IRS proteins (Yenush, 1996). Furthermore, genetic evidence in Drosophila suggests that InR can signal in the absence of Chico, the IRS1-4 homolog. In order to understand the molecular basis for differences in strength of InR phenotypes, the cytoplasmic region of several InR alleles was sequenced. In the cytoplasmic portion, 5 out of 22 alleles carry a point mutation. All of them map to conserved amino acid residues within the kinase domain. Two of these point mutations lead to premature stop codons and three are missense mutations. In humans, most of the mutations that occur within the tyrosine kinase domain of the Inr have been shown to impair insulin-stimulated tyrosine kinase activity. InR353 (Arg1419Cys) affects an active site residue, which mediates insulin receptor kinase substrate specificity. Remarkably, a human patient with severe growth retardation associated with insulin resistance, a syndrome called leprechaunism, carries an amino acid exchange at the corresponding position (Arg1092Glu). It is the only reported homozygous viable mutation in the kinase domain of the human Inr. The patient's parents were heterozygous for this substitution and had severe insulin resistance, but no growth anomalies. Similarly, heterozygosity for InR alleles does not lead to growth phenotypes. These results suggest a role for the insulin receptor in growth control that has been conserved from insects to humans (Brogiolo, 2001).

It has been proposed that a bona fide growth control gene should meet two criteria, namely that elimination should result in growth retardation, whereas overexpression of the gene should promote excessive growth. To determine whether InR has a direct growth- and proliferation-promoting effect, a wild-type InR cDNA was overexpressed using the UAS/Gal4 system. Expressing UAS-InRwt specifically in proliferating eye precursor cells using an eyeless-Gal4 driver results in a dramatic outgrowth in the adult eye because of an increase in the number of ommatidia. Histological sections through the overgrown eyes reveal essentially normal cell differentiation but a slight increase in the size of photoreceptor cell bodies. To further explore the effect on cell size, InR was overexpressed in clones of cells during cell differentiation. External observation of such clones shows strongly enlarged ommatidia. Histological sections reveal a cell-autonomous increase in photoreceptor cell size but only a moderate disruption of the ommatidial pattern. Taken together, these results indicate that InR activity controls growth in two ways: by regulating both cell proliferation and cell size. Interestingly, although overexpression of Dp110 has been shown to increase cell size, it does not increase cell division rates. The IRS homolog Chico contains consensus binding sites for the Drk/Grb2 adaptor and thus may provide a link to the Ras/MAPK pathway. Activation of InR may promote cell growth and cell division by activation of two signaling pathways. Indeed, MAPK activation is observed in extracts of heads overexpressing an activated form of InR (Brogiolo, 2001).

To identify extracellular ligands that regulate InR activity during development, a search of the Drosophila genome was carried out, looking for genes encoding insulin-like peptides. Using the conserved spacing of four cysteines within the A chain as a signature for insulin-like peptides, seven predicted genes matching these criteria were identified, and termed dilp1-7, for Drosophila insulin-like peptides (DILP). dilp1-5 are on the third chromosome at cytological position 67C1-2, and constitute a cluster of four contiguous insulin-related genes, with dilp5 separated by one intervening gene from dilp4. The other genes, dilp6 and dilp7, are on the X chromosome at two different loci at cytological positions 2F4 and 3F2, respectively. dilp1-7 encode putative precursor proteins of 107 to 156 amino acid residues in length that are structurally similar to preproinsulin, with a signal peptide, a B chain, a C peptide, and an A chain. Consensus cleavage sites between the B and A chains of all seven DILPs suggest that the active peptides consist of two separate polypeptide chains. Thus, these peptides resemble insulin rather than IGF1 or IGF2, which are single polypeptides. Comparison of the amino acid sequence of the A and B chains of DILP1-7 with insulin, IGF1, and IGF2 again reveals a higher degree of identical amino acids between these peptides and insulin. DILP2 is the most closely related, with 35% identity to mature insulin. These structural similarities suggest that DILP1-7 are candidate ligands for InR (Brogiolo, 2001).

To determine the expression pattern of the insulin-like genes, in situ hybridization was performed on embryos and larval tissues. In the embryo, only dilp2, dilp4, and dilp7 are expressed at different levels in the mesoderm and midgut. It is interesting to note that the main insulin-producing organs in mammals, the Langerhans islets in the pancreas, are of endodermal origin. Four of the seven genes show a remarkably specific and unique pattern of expression in larvae. dilp2, dilp3, and dilp5 display high expression levels in seven cells of anteromedial localization in the brain hemispheres that may correspond to neurosecretory cells. dilp3 is exclusively transcribed in these seven cells during larval development, whereas dilp2 and dilp5 show additional expression domains. dilp7 mRNA detection is restricted to the ventral nerve cord in a segmental fashion; in four pairs of ventrally located cells in the most posterior abdominal segments, and in one pair of dorsally located cells in A1 or A2. Interestingly, none of the dilps shows detectable levels of expression in the larval fat body (Brogiolo, 2001).

Expression of insulin-related genes in neurosecretory cells has been identified in other invertebrates, such as the insects Bombyx mori and Locusta migratoria and in the mollusc Lymnaea stagnalis (Kawakami, 1989, Hetru, 1991 and Smit, 1988). In Bombyx mori, the neurosecretory cells in the brain are connected to the corpora cardiaca, a secretory gland from which release of insulin-like hormones is triggered by nutrient levels (possibly carbohydrate levels) (Masumura, 2000). It is speculated that DILP-expressing neurosecretory cells are connected to the ring gland (the compound endocrine gland of Drosophila), which includes the cells of the corpora cardiaca. Release of DILPs from the ring gland may also be under nutritional control. The complex expression pattern of the DILPs, however, suggests a combination of neurosecretory and autocrine/paracrine control mechanisms of cell growth and division during larval development. Mutations in individual dilp genes or targeted ablation of specific DILP-expressing cells may help resolve the functions of the Drosophila insulins (Brogiolo, 2001).

To gain insight into the function of the DILPs, one insulin-like peptide was overexpressed. For this purpose, DILP2 was chosen, because it is the closest homolog of human insulin and because it is the only DILP with broad expression in imaginal discs. If DILP2 is a limiting ligand of InR, it would be expected that overexpression of DILP2 should promote growth. Indeed, repeated induction of ubiquitous expression of DILP2 during development by means of the UAS/Gal4 system gives rise to bigger flies (39% increase in body weight). Analysis of the eyes of such flies reveals an increase in the number of ommatidia (from 733+/-10 to 767+/-25 in male flies). Furthermore, quantitative analysis of the wing blade shows an increase in both cell size (by 9%) and cell number (by 11%). These results suggest a role for DILP2 in controlling organism size by augmenting both the cell number and cell size of different organs (Brogiolo, 2001).

In humans, the in vivo role of insulin as a growth factor is inferred from clinical syndromes, in which excessive insulin secretion results in excessive growth and where a severe deficiency of insulin secretion is associated with poor intrauterine and postnatal growth. For instance, neonates born to women with diabetes in pregnancy or born with Beckwith-Wiedemann syndrome or Nesidioblastosis are macrosomic. In all cases, the growth anomaly is associated with hyperinsulinemia during embryonic development. The demonstration in transgenic flies that overexpression of an insulin-like peptide during development can increase animal size provides further evidence for an evolutionarily conserved role of the insulin pathway in growth control (Brogiolo, 2001).

The complementarity between the loss-of-function phenotype of InR and the DILP2 overexpression phenotype (increase in size) suggests that DILP2 may be one of the ligands for InR. A deficiency [Df(3L)AC1] uncovering dilp1-5 was found to dominantly suppressed the big and rough eye phenotype caused by targeted overexpression of InR in differentiating eye cells. To test whether the observed dominant suppression is caused by hemizygosity for dilp2, the dilp2 gene dosage was selectively increased by crossing in the UAS-dilp2 transgene. A single copy of UAS-dilp2 is sufficient to revert the suppression by Df(3L)AC1, strongly suggesting that dilp2 is rate limiting for the InR overexpression phenotype. An analysis of individually mutated dilp genes will be required to determine the contribution of the other dilps of the cluster (dilp13-5) to the suppressive effect of Df(3L)AC1 (Brogiolo, 2001).

To examine whether InR is limiting for the DILP2 overexpression phenotype, InR activity was lowered in a DILP2-overexpressing background. Indeed, introducing one mutant copy of InR (InR304) dominantly reduces the increased body weight, cell size, and cell number caused by ubiquitous DILP2 overexpression, indicating a strong genetic interaction between InR and dilp2. Persistent expression of DILP2 under the control of an actin promoter (Act5C-Gal4) causes embryonic lethality. This lethality is dependent on normal levels of InR, since expression of DILP2 in the presence of strongly reduced levels of InR generate viable adults that are small and developmentally delayed. These results are consistent with InR mediating the effects of DILP2. Furthermore, given that a viable heteroallelic combination of PKB alleles is also able to suppress the embryonic lethal phenotype of DILP2 overexpression, it is postulated that the action of DILP2 by InR is transduced at least in part through the Chico/Pi(3)K/PKB pathway (Brogiolo, 2001).

In humans, syndromes with mutations in the insulin receptor or with excessive insulin secretion lead to growth abnormalities. This study shows in vivo that altering expression levels of a Drosophila insulin-like gene and varying the activity of the Drosophila insulin receptor changes the size and number of cells in organs, thereby regulating organism size. It seems, therefore, that the insulin receptor pathway has been conserved during evolution for a role in growth control from insects to humans. Given the highly tissue-specific expression of the dilps in the central nervous system and a broad expression in precursor tissues of adult organs, a nutritionally regulated mechanism is proposed whereby Drosophila insulin-like peptides coordinate growth in a neurosecretory and local fashion (Brogiolo, 2001).

Transformed Drosophila cells evade diet-mediated insulin resistance through wingless signaling

Cancer cells demand excessive nutrients to support their proliferation but how cancer cells sense and promote growth in the nutrient favorable conditions remain incompletely understood. Epidemiological studies have indicated that obesity is a risk factor for various types of cancers. Feeding Drosophila a high dietary sugar was previously demonstrated to not only direct metabolic defects including obesity and organismal insulin resistance, but also transform Ras/Src-activated cells into aggressive tumors. This study demonstrates that Ras/Src-activated cells are sensitive to perturbations in the Hippo signaling pathway. Evidence that nutritional cues activate Salt-inducible kinase, leading to Hippo pathway downregulation in Ras/Src-activated cells. The result is Yorkie-dependent increase in Wingless signaling, a key mediator that promotes diet-enhanced Ras/Src-tumorigenesis in an otherwise insulin-resistant environment. Through this mechanism, Ras/Src-activated cells are positioned to efficiently respond to nutritional signals and ensure tumor growth upon nutrient rich condition including obesity (Hirabayashi, 2015).

The prevalence of obesity is increasing globally. Obesity impacts whole-body homeostasis and is a risk factor for severe health complications including type 2 diabetes and cardiovascular disease. Accumulating epidemiological evidence indicates that obesity also leads to elevated risk of developing several types of cancers. However, the mechanisms that link obesity and cancer remain incompletely understood. Using Drosophila, a whole-animal model system has been developed to study the link between diet-induced obesity and cancer: this model has provided a potential explanation for how obese and insulin resistant animals are at increased risk for tumor progression (Hirabayashi, 2015).

Drosophila fed a diet containing high levels of sucrose (high dietary sucrose or ‘HDS') developed sugar-dependent metabolic defects including accumulation of fat (obesity), organismal insulin resistance, hyperglycemia, hyperinsulinemia, heart defects and liver (fat body) dysfunctions. Inducing activation of oncogenic Ras and Src together in the Drosophila eye epithelia led to development of small benign tumors within the eye epithelia. Feeding animals HDS transformed Ras/Src-activated cells from benign tumor growths to aggressive tumor overgrowth with tumors spread into other regions of the body (Hirabayashi, 2013). While most tissues of animals fed HDS displayed insulin resistance, Ras/Src-activated tumors retained insulin pathway sensitivity and exhibited an increased ability to import glucose. This is reflected by increased expression of the Insulin Receptor (InR), which was activated through an increase in canonical Wingless (Wg)/dWnt signaling that resulted in evasion of diet-mediated insulin resistance in Ras/Src-activated cells. Conversely, expressing a constitutively active isoform of the Insulin Receptor in Ras/Src-activated cells (InR/Ras/Src) was sufficient to elevate Wg signaling, promoting tumor overgrowth in animals fed a control diet. These results revealed a circuit with a feed-forward mechanism that directs elevated Wg signaling and InR expression specifically in Ras/Src-activated cells. Through this circuit, mitogenic effects of insulin are not only preserved but are enhanced in Ras/Src-activated cells in the presence of organismal insulin resistance (Hirabayashi, 2015).

These studies provide an outline for a new mechanism by which tumors evade insulin resistance, but several questions remain: (1) how Ras/Src-activated cells sense the organism's increased insulin levels, (2) how nutrient availability is converted into growth signals, and (3) the trigger for increased Wg protein levels, a key mediator that promotes evasion of insulin resistance and enhanced Ras/Src-tumorigenesis consequent to HDS. This study identifies the Hippo pathway effector Yorkie (Yki) as a primary source of increased Wg expression in diet-enhanced Ras/Src-tumors. Ras/Src-activated cells are sensitized to Hippo signaling, and even a mild perturbation in upstream Hippo pathway is sufficient to dominantly promote Ras/Src-tumor growth. Functional evidence is provided that increased insulin signaling promotes Salt-inducible kinases (SIKs) activity in Ras/Src-activated cells, revealing a SIKs-Yki-Wg axis as a key mediator of diet-enhanced Ras/Src-tumorigenesis. Through this pathway, Hippo-sensitized Ras/Src-activated cells are positioned to efficiently respond to insulin signals and promote tumor overgrowth. These mechanisms act as a feed-forward cassette that promotes tumor progression in dietary rich conditions, evading an otherwise insulin resistant state (Hirabayashi, 2015).

Previously work has demonstrated that Ras/Src-activated cells preserve mitogenic effects of insulin under the systemic insulin resistance induced by HDS-feeding of Drosophila (Hirabayashi, 2013). Evasion of insulin resistance in Ras/Src-activated cells is a consequence of a Wg-dependent increase in InR gene expression (Hirabayashi, 2013). This study identified the Hippo pathway effector Yki as a primary source of the Wnt ortholog Wg in diet-enhanced Ras/Src-tumors. Mechanistically, functional evidence is provided that activation of SIKs promotes Yki-dependent Wg-activation and reveal a SIK-Yki-Wg-InR axis as a key feed-forward signaling pathway that underlies evasion of insulin resistance and promotion of tumor growth in diet-enhanced Ras/Src-tumors (Hirabayashi, 2015).

In animals fed a control diet, at most a mild increase was observed in Yki reporter activity within ras1G12V;csk-/- cells. A previous report indicates that activation of oncogenic Ras (ras1G12V) led to slight activation of Yki in eye tissue. Activation of Src through over-expression of the Drosophila Src ortholog Src64B has been shown to induce autonomous and non-autonomous activation of Yki. In contrast, inducing activation of Src through loss of csk (csk-/-) failed to elevate diap1 expression. The results indicate that activation of Yki is an emergent property of activating Ras plus Src (ras1G12V;csk-/-). However, this level of Yki-activation was not sufficient to promote stable tumor growth of Ras/Src-activated cells in the context of a control diet: Ras/Src-activated cells were progressively eliminated from the eye tissue (Hirabayashi, 2013). It was, however, sufficient to sensitize Ras/Src-activated cells to upstream Hippo pathway signals: loss of a genetic copy of ex-which was not sufficient to promote growth by itself-dominantly promoted tumor growth of Ras/Src-activated cells even in animals fed a control diet. These data provide compelling evidence that Ras/Src-transformed cells are sensitive to upstream Hippo signals (Hirabayashi, 2015).

SIK was recently demonstrated to phosphorylate Sav at Serine-413, resulting in dissociation of the Hippo complex and activation of Yki (Wehr, 2013). SIKs are required for diet-enhanced Ras/Src-tumor growth in HDS. Conversely, expression of a constitutively activated isoform of SIK was sufficient to promote Ras/Src-tumor overgrowth even in a control diet. Mammalian SIKs are regulated by glucose and by insulin signaling. However, a recent report indicated that glucagon but not insulin regulates SIK2 activity in the liver. The current data demonstrate that increased insulin signaling is sufficient to promote SIK activity through Akt in Ras/Src-activated cells. It is concluded that SIKs couple nutrient (insulin) availability to Yki-mediated evasion of insulin resistance and tumor growth, ensuring Ras/Src-tumor growth under nutrient favorable conditions (Hirabayashi, 2015).

The results place SIKs as key sensors of nutrient and energy availability in Ras/Src-tumors through increased insulin signaling and, hence, increased glucose availability. SIK activity promotes Ras/Src-activated cells to efficiently respond to upstream Hippo signals, ensuring tumor overgrowth in organisms that are otherwise insulin resistant. One interesting question is whether this mechanism is relevant beyond the context of an obesity-cancer connection: both Ras and Src have pleiotropic effects on developmental processes including survival, proliferation, morphogenesis, differentiation, and invasion, and these mechanisms may facilitate these processes under nutrient favorable conditions. From a treatment perspective the current data highlight SIKs as potential therapeutic targets. Limiting SIK activity through compounds such as HG-9-91-01 may break the connection between oncogenes and diet, targeting key aspects of tumor progression that are enhanced in obese individuals (Hirabayashi, 2015).

Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila

Starvation induces sustained increase in locomotion, which facilitates food localization and acquisition and hence composes an important aspect of food-seeking behavior. This study investigated how nutritional states modulate starvation-induced hyperactivity in adult Drosophila. The receptor of adipokinetic hormone (AKHR), the insect analog of glucagon, is required for starvation-induced hyperactivity. AKHR is expressed in a small group of octopaminergic neurons in the brain. Silencing AKHR+ neurons and blocking octopamine signaling in these neurons eliminates starvation-induced hyperactivity, whereas activation of these neurons accelerates the onset of hyperactivity upon starvation. Neither AKHR nor AKHR+ neurons are involved in increased food consumption upon starvation, suggesting that starvation-induced hyperactivity and food consumption are independently regulated. Single cell analysis of AKHR+ neurons identified the co-expression of Drosophila insulin-like receptor (dInR), which imposes suppressive effect on starvation-induced hyperactivity. Therefore, insulin and glucagon signaling exert opposite effects on starvation-induced hyperactivity via a common neural target in Drosophila (Yu, 2016).

Food seeking and food consumption are essential for the acquisition of food sources, and hence survival, growth, and reproduction of animal species. Starvation influences food-seeking behavior via both modulating the perception of food cues as well as enhancing flies' locomotor activity. Accumulated evidence has suggested that starvation modulates the activity of ORNs via multiple neural and hormonal cues, which in turn facilitates odor driven food search and food consumption. Similarly, starvation also modulates the perception of food taste via the relative sensitivity of appetitive sweet-sensing and aversive bitter-sensing GRNs,which may in turn increase the attractiveness of food taste. However, how starvation increases the locomotor activity of flies remains largely uncharacterized (Yu, 2016).

Consistent with previous reports, this study has shown that starved fruit flies exhibit sustained increase in their locomotor activity, which can be suppressed by food consumption induced by both nutritive and non-nutritive food cues. The present study has shown that a small group of neurons located in the subesophageal zone (SEZ) region of the fly brain are both necessary and sufficient for starvation induced hyperactivity. These neurons sense the changes in flies' internal nutritional states by directly responding to two sets of hormones, AKH and DILPs, and modulate locomotor activity in response. Single cell analysis has identified that these AKHR+dInR+ neurons are octopaminergic, which offers an entry point to trace the downstream neural circuitry that regulates starvation-induced hyperactivity. For example, there are seven candidate octopamine receptors in fruit flies and it would be of interest to investigate whether any of these receptors and the receptor-expressing neurons are involved in locomotor regulation upon starvation (Yu, 2016).

AKH and DILPs are two sets of functionally counteracting hormones in fruit flies. As its mammalian analog glucagon, the reduction in circulating sugars induces the release of AKH, which in turn mobilizes fat storage and provides energy supply for flies. In contrast, DILPs, the insect analog of mammalian insulin, function as satiety hormones. Dietary nutrient induces the release of DILPs into the hemolymph, which in turn promotes protein synthesis, body growth, and other anabolic processes. This study has shown that these two hormonal signaling systems exert opposite effects on starvation-induced hyperactivity via a small group of AKHR+InR+ octopaminergic neurons. These results suggest that these AKHR+dInR+ neurons can integrate the inputs from the two hormonal signaling systems representing hunger and satiety at the same time, and modulate flies' locomotor activity. This elegant yet concise design allows these neurons to be responsive to rapid changes in the internal nutritional states as well as food availability. Furthermore, it is possible that besides hunger and satiety, other physiological states such as wakefulness, stress, and emotions also influence flies' locomotor activity. Notably, single cell analysis has shown that these AKHR+dInR+ neurons also sparsely express other neuropeptide receptors, suggesting that at least small portions of these neurons may also receive input from other neuropeptidergic systems (Yu, 2016).

Starved animals exhibited increased locomotion and food consumption, the transition of which relies on the detection of food cues. But whether these two behaviors are interdependently or independently regulated remains unclear. This study has shown that these two behaviors are dissociable from each other in fruit flies. On the one hand, although AKHR+ neurons exert robust modulatory effect on starvation-induced hyperactivity, these neurons are neither necessary nor sufficient for starvation-induced food consumption. On the other hand, the regulation of food consumption is independent of starvation-induced hyperactivity as well. Previous studies have shown that a small subset of GABAergic neurons in the fly brain regulates food consumption but exerts no effect on 10 starvation-induced hyperactivity (Pool, 2014). In addition, several neuropeptides are known to regulate food consumption, such as Hugin, NPF, sNPF, Leucokinin, and AstA. However this study found in an RNAi screen that the receptors of these neuropeptides were not involved in the regulation of starvation-induced hyperactivity. Taken together, it is likely that starvation-induced hyperactivity and food consumption are independently regulated by different sets of hormonal cues, and that AKHR+ neurons are only involved in the former but not the latter. These results may shed light on the regulation of food intake in mammals, especially whether starvation-induced hyperactivity and food consumption are also independently regulated by different sets of hormones and distinct neural circuitry in mammals (Yu, 2016).



The Drosophila Insulin receptor (InR) regulates cell growth and proliferation through the PI3K/Akt pathway, which is conserved in metazoan organisms. The Drosophila forkhead-related transcription factor Foxo is a key component of the insulin signaling cascade. Foxo is phosphorylated by Akt upon insulin treatment, leading to cytoplasmic retention and inhibition of its transcriptional activity. Mutant Foxo lacking Akt phosphorylation sites no longer responds to insulin inhibition, remains in the nucleus, and is constitutively active. Foxo activation in S2 cells induces growth arrest and activates two key players of the InR/PI3K/Akt pathway: the translational regulator d4EBP/Thor (eukaryotic initiation factor 4E binding protein) and the InR itself. Induction of d4EBP likely leads to growth inhibition by Foxo, whereas activation of InR provides a novel transcriptionally induced feedback control mechanism. Targeted expression of Foxo in fly tissues regulates organ size by specifying cell number with no effect on cell size. These results establish Foxo as a key transcriptional regulator of the insulin pathway that modulates growth and proliferation (Puig, 2003).

Foxo regulates cell cycle arrest possibly by transcriptionally activating genes implicated in cell division or in cell growth. As an initial attempt to identify target genes of Foxo, DNA microarrays were used to assess gene expression profiles in S2 cells stably transfected with mutant Foxo and grown in the presence of insulin. Cells expressing wild-type Foxo or untransfected S2 cells subjected to the same treatment were assayed as controls (Puig, 2003).

Two-hundered and seventy-seven genes were found to be up-regulated in Foxoa3-expressing cells when compared with Foxo-expressing cells or untransfected S2 cells. Interestingly, two genes that were consistently and specifically up-regulated in these conditions were the Drosophila InR gene (13.5-fold) and the Drosophila 4EBP gene (25-fold). Both genes have been implicated in the regulation of cell growth by insulin. To confirm that InR and 4EBP are bona fide transcriptional targets of Foxo, the same experiment described above was performed but in the presence of cycloheximide to inhibit translation. As expected, both InR and 4EBP continue to be transcriptionally activated (2.5- and 3.1-fold, respectively) by FOXOA3 but not Foxo in the insulin-repressed state. This result suggests that Foxo, when released from control by the insulin/dAkt cascade, is involved in transcription from the InR and 4EBP promoters (Puig, 2003).

To confirm these microarray results and to independently quantitate the increase in mRNA transcription, RNase protection assays were performed with mRNAs extracted from cells stably transfected with either Foxo or FoxoA3. Indeed, FoxoA3 stimulates transcription of Drosophila 4EBP and InR by 16.3- and 11-fold, respectively. A time-course experiment confirmed that Drosophila InR mRNA increases rapidly upon FoxoA3 expression: 3 h after CuSO4 addition, there is already an 8-fold increase, reaching 20-fold after 9 h of CuSO4 induction. Similar results were obtained for Drosophila 4EBP. These experiments suggest that Foxo expression specifically activates both Drosophila InR and 4EBP transcription, thus unmasking an important feedback control mechanism in this pathway involving Foxo and InR (Puig, 2003).

Having obtained evidence that exogenously transfected Foxo responds to insulin and regulates both the downstream target gene 4EBP and the feedback control target InR, it was of interest to know if endogenous Foxo would also activate transcription of these genes. The PI3K inhibitor LY294002 was used to activate endogenous Foxo or insulin to deactivate it. S2 cells grown in the absence of serum for 48 h were treated either with LY294002 or insulin. Total RNA was extracted and RNase protection was performed to detect Drosophila InR and 4EBP mRNAs. Both mRNA levels are significantly increased after LY294002 treatment (5.3-fold for dInR and 4-fold for d4EBP) when compared with insulin treatment. This result provides further evidence indicating that the PI3K–Akt pathway regulates InR and 4EBP transcription via Foxo (Puig, 2003).

It was of interest to determine whether Foxo directly binds to the promoters of Drosophila 4EBP and InR. To identify the DNA region recognized by Foxo in these two promoters, a 1708-bp fragment of the 4EBP promoter and a 1562-bp fragment of the InR promoter were inserted into a luciferase reporter vector. When transfected into S2 cells, these fragments responded to Foxo activation (3-fold for 4EBP, >200-fold for InR. A series of deletions lacking upstream sequences still responded to Foxo activation, albeit more weakly, suggesting that Foxo can bind the DNA in a region close to the start of transcription (485 bp for the d4EBP promoter and 194 bp for the dInR promoter). In contrast, Foxo completely fails to activate a reporter construct in which upstream activating sequences (UAS) for the transcription factor GAL4 are fused to the luciferase gene, confirming that transcription activation is specific for both 4EBP and InR promoters (Puig, 2003).

Interestingly, 125 bp upstream of the transcription start site of the d4EBP promoter there are three tandem copies of a putative FOXO4 recognition element (FRE). These elements are reminiscent of the ones present in the human glucose-6-phosphatase promoter, previously shown to bind FOXO4 (Yang, 2002). This was reassuring because Foxo and FOXO4 share 85% identity in the core of the forkhead DNA-binding domain. Similarly, several putative FRE sequences appear in the InR promoter in the region comprising nucleotides -1434 to -70 (Puig, 2003).

To determine whether Foxo binds these putative FREs, band shift experiments were performed with a 113-bp DNA probe encompassing the 4EBP FRE motifs and with 12 separate DNA probes (ranging from 100 to 150 bp) spanning a region of 1.4 kb from the InR promoter. Purified recombinant Foxo expressed in Escherichia coli efficiently binds the 113-bp FRE-containing fragment from the 4EBP promoter compared with control DNA fragments. Furthermore, Foxo binding to the 4EBP promoter fragment can be efficiently competed with an unlabeled 113-bp 4EBP promoter fragment but not with nonspecific DNA. Similarly, purified recombinant Foxo binds efficiently to 5 out of 12 of the DNA fragments located within the InR promoter. As expected, each of the five DNA fragments bound by Foxo contains putative FREs. Thus, Foxo can specifically bind to both promoters in vitro. To determine whether Foxo also binds these same DNA regions in vivo, chromatin immunoprecipitation (ChIP) experiments were performed with S2 cells expressing either Foxo or dFoxoA3. Cells were incubated with serum, and Foxo expression was induced with the addition of CuSO4. After 6 h, cells were cross-linked with formaldehyde, and extracts were prepared and immunoprecipitated. After reversal of cross-links, DNA was recovered, and PCR was performed with primers encompassing regions containing putative FREs in both promoters. The results indicate that Foxo can directly bind to both the 4EBP and InR promoters in vivo. These results establish that Foxo can specifically bind the 4EBP and InR promoters both in vitro and in vivo (Puig, 2003).

To demonstrate that Foxo can directly activate transcription of these promoters in vitro, the constructs were used that contain 485 bp of the 4EBP promoter region and 514 bp of the InR promoter region, respectively. Addition of purified recombinant Foxo to in vitro reactions activates transcription of these promoters by at least 3-fold (4EBP) and 5.5-fold (InR), which is comparable to the activation observed in vivo. Under in vitro transcription conditions, activation of the 4EBP promoter by Foxo becomes rapidly saturated with increasing amounts of Foxo. As expected, Foxo also activates (up to sixfold) a synthetic promoter bearing four FOXO4-binding sites placed upstream of the alcohol dehydrogenase distal promoter. Together these results show that transcription of 4EBP and InR can be directly activated by Foxo in vitro (Puig, 2003).

Insulin-like receptor and nutritional status

Studies in Drosophila have characterized Insulin receptor/Phosphoinositide 3-kinase (Inr/PI3K) signaling as a potent regulator of cell growth, but an understanding of its function during development has remained uncertain. Inhibiting Inr/PI3K signaling phenocopies the cellular and organismal effects of starvation, whereas activating this pathway bypasses the nutritional requirement for cell growth, causing starvation sensitivity at the organismal level. Consistent with these findings, studies using a pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion as an indicator for PI3K activity show that PI3K is regulated by the availability of dietary protein in vivo. It is surmised that an essential function of insulin/PI3K signaling in Drosophila is to coordinate cellular metabolism with nutritional conditions (Britton, 2002).

To test whether PI3K is required for larval growth, its activity was inhibited by expressing p60, Deltap60, or Pten in large domains of the larva using the Gal4/UAS system. p60 is an adaptor that couples Inr to Pi3K92E (Dp110), and Deltap60 is a deletion variant lacking part of the Dp110 binding domain. These molecules and their mammalian homologs have dominant-negative effects on PI3K activity when overexpressed, presumably because they compete with endogenous Dp110/p60 complexes for binding sites on upstream activators such as insulin receptors and IRSs. When p60 is expressed under the control of Act-Gal4 (expressed ubiquitously) or Adh-Gal4 (expressed predominantly in fat body), larvae remain growth arrested in the first instar for as long as 2 weeks. Deltap60 and Pten had similar effects. Dissection of these animals revealed that all of their tissues and organs were proportionally reduced. This developmental arrest is indistinguishable from the effects of starvation or inhibition of protein synthesis (Britton, 2002).

To test whether PI3K is autonomously required for growth in the different larval cell types, the Flp/Gal4 technique was used to express p60, Deltap60, or Pten in scattered cells throughout the larva. This method employs the Act>CD2>Gal4 and hs-Flp transgenes to activate UAS-linked target genes, including the cell marker UAS-GFPnls, in cell clones. Heat shock-independent activation of Gal4 occurs prior to the onset of larval growth and DNA endoreplication in 1%-10% of cells (depending on organ) in the fat body, gut, salivary glands, renal (Malpigian) tubules, and epidermis. Cells overexpressing p60, Deltap60, or Pten in the salivary glands and fat body are greatly reduced in size and have much smaller nuclei with far less DNA than adjacent control cells. Similar effects are observed in other larval tissues. Despite their reduced growth, p60-, Deltap60-, and Pten-expressing cells are found at approximately the same frequencies as control GFP-marked cells. Apoptotic cells are not observed. Thus, reductions in PI3K activity are not incompatible with cell viability. Overt effects on cell morphology that might reflect changes in cell adhesion, motility, or identity are also not observed. It is concluded that reducing Inr/PI3K activity in the differentiated tissues of the larva has cell-autonomous effects that are limited to reducing cell growth and DNA replication (Britton, 2002).

These observations suggest that PI3K activity might be responsive to nutritional conditions. To directly test this possibility, a fusion protein was made for use as an in vivo reporter for PI3K activity. The pleckstrin homology (PH) domain of the Drosophila homolog of general receptor for phosphoinositides-1 (GRP1) was fused to green fluorescent protein (GFP), generating a protein called GPH (GFP-PH domain). PH domains from mammalian GRP1 genes bind specifically to phosphatidylinositol-3,4,5-P3 (PIP3), the second messenger generated by class I PI3-kinases. Since PIP3 generally resides in lipid membranes, particularly the plasma membrane, GRP1 is recruited to membranes when PI3-kinase activity raises cellular levels of PIP3. Fusion proteins containing the GRP1 PH domain are likewise recruited to plasma membranes by binding PIP3, and thus serve as in situ reporters for PI3K activity (Britton, 2002).

For in vivo studies, the GPH gene was placed under control of the Drosophila ß-tubulin promotor, generating a gene called 'tGPH' (tubulin-GPH), and introduced into Drosophila by P element-mediated transformation. Membrane localization of tGPH was observed in the larval epidermis, fat body, salivary glands, malpighian tubules, and wing imaginal discs. Cytoplasmic and nuclear tGPH was also visible in these cell types. The degree of membrane localization depends upon the developmental stage. Epidermal cells show little membrane localization of tGPH in embryos or newly hatched first instar (L1) larvae, but have strong membrane localization in second (L2) and early third (L3) instar larvae. Later, in wandering stage L3 larvae, membrane-associated tGPH is again diminished. Similar trends are observed in the fat body. These variations might reflect changes in the levels of Inr, PI3K, Pten, or insulin-like peptides (dILPs) in the larva as it feeds and grows (Britton, 2002).

To test whether tGPH localization was responsive to PI3K activity in vivo, Inr or Dp110 was overexpressed using the Gal4 system. Either gene causes a striking redistribution of tGPH to plasma membranes in cells of the fat body, epidermis, Malpighian tubules, gut, and imaginal discs. To determine whether endogenous PI3K is responsible for the membrane localization of tGPH, PI3K activity was suppressed by expressing p60 with the heat-inducible driver hs-Gal4. In the larval epidermis, partial loss of membrane-bound tGPH is apparent 1.5 hr post-heat shock (phs), and by 4 hr, phs tGPH is nearly completely lost from cell membranes. Similar results were obtained in the fat body when either p60 or dPten were expressed mosaically using the Flp/Gal4 method (Britton, 2002).

To determine whether PI3K activity is nutritionally modulated, tGPH localization was monitored after starvation. Early L2 larvae (48-60 hr AED) were deprived of dietary protein by culture on either 20% sucrose or Sang's defined media lacking casein. These treatments arrest cell growth in all of the differentiated larval tissues. L2 larvae fed on either protein-free diet survive for up to 14 days, but a marked shrinkage of cells in the epidermis and fat body is observed. In the epidermis of early L2 larvae, culture on either protein-free diet causes membrane-bound tGPH to be diminished after 24 hr, and to be nearly undetectable after 48 hr. Levels of total tGPH also decrease after protein deprivation, but nuclear and cytoplasmic tGPH remain detectable for more than 6 days. Loss of membrane-associated tGPH also occurs in fat body cells of protein-deprived L2 larvae, with similar kinetics. Starvation causes shrinkage of the nuclei and nonlipid cytoplasm in fat body cells, leaving large tGPH-negative lipid droplets that occupy most of the cell volume. To test whether membrane association of tGPH is reversible, L2 larvae were cultured on 20% sucrose for 8 days and then returned to whole food. In this experiment, tGPH levels rose and the protein reassociated with plasma membranes in epidermal cells between 24 and 48 hr after feeding. These results indicate that cellular levels of PIP3 drop as a consequence of starvation for dietary protein (Britton, 2002).

Terminally differentiated endoreplicating tissues (ERTs) constitute most of a Drosophila larva, and the growth of these tissues accounts for virtually all of the ~200-fold mass increase sustained by the animal during the larval stages. During larval life, the ERTs provide a physiologically nurturing environment for undifferentiated imaginal cells and neuroblasts, which generate much of the reproductive adult stage. Most of the biomass accumulated in the ERTs is eventually recycled into these progenitor cells as they form the adult. This study provides evidence that Inr/PI3K signaling coordinates nutritional status with ERT cell metabolism and growth. To determine whether Inr/PI3K signaling can maintain cell growth in the face of starvation, Flp/Gal4 was used to express Dp110 or Inr in scattered ERT cells, and then changes in cell size and DNA replication were assessed at time points during a protein starvation regime. At larval hatching, prior to starvation, cells expressing Dp110 or Inr in the gut, fat body, Malpighian tubules, and epidermis are only slightly larger than nonexpressing cells. After several days of starvation on 20% sucrose, Inr- or Dp110-expressing cells in these organs are much larger than adjacent control cells, and have visibly increased DNA content. BrdU incorporation indicated that gut and fat body cells expressing Dp110 or Inr continue to replicate their DNA for at least 2 to 3 days under starvation conditions. Normally, DNA endoreplication in these cells ceases within 1 to 2 days of starvation (Britton, 1998). A catalytically inactive PI3K, Dp110D945A, does not promote cell growth or DNA endoreplication, indicating that lipid kinase activity was required. These experiments indicate that active Inr/PI3K signaling is sufficient to bypass the nutritional requirement for cellular growth and DNA replication in many larval cell types, and that this effect is cell autonomous (Britton, 2002).

To explore the means by which Inr/PI3K signaling induces cell growth, an examination was carried out of the morphology of fat body cells in which PI3K activity had been manipulated. Fat body cells accumulate large stores of protein, carbohydrate, and lipids during larval life, and also produce growth factors. During the third larval instar, these accumulations of nutrients cause fat body cells to become opaque. These nutrients are normally utilized during metamorphosis, but if a larva is starved, they are precociously mobilized into the haemolymph to support the animal during the ensuing dietary crisis. This causes the fat body cells to shrink and become clear as they lose organelles by autophagy and deplete stored metabolites (Britton, 2002).

Expression of Inr or PI3K in fat body cells increases the opacity of the cytoplasm, and thus promotes nutrient storage. A similar cytoplasmic effect is observed in intestinal cells from L1 animals. Close inspection has revealed that in both cell types, ectopic Inr or PI3K decreases the size of prominent vesicles in the cytoplasm. Induction of p60 in early L3 larvae has opposite effects, causing fat body cells to become more translucent. A loss of opacity was observed in fat bodies from Dp110 mutants after their growth arrest at L3. In further tests, fat body cells were stained for lipids with Nile red or for protein with Texas red X succinimidyl ester. This revealed that the cytoplasm of Inr-expressing cells contains many more, but much smaller, lipid droplets than neighboring control cells. In summary, activation of the Inr/PI3K pathway has profound effects on cytoplasmic composition. These effects mimic changes in the fat body that normally take place late in the L3 stage when nutrient storage by these cells is maximal. Suppression of Inr/PI3K activity has opposite effects on cytoplasmic composition, and these appear to mimic the mobilization of nutrients that normally accompanies starvation (Britton, 2002).

Considering the above results, it should be advantageous to larvae to downregulate insulin/PI3K signaling when nutrients are limited, since this would suppress nutrient storage and cell growth and allow nutrient mobilization by tissues such as the fat body. This idea was tested by hyperactivating Inr/PI3K signaling and then tracking development under different nutritional conditions. Several Gal4 drivers were used to induce expression in large numbers of cells, including Adh-Gal4 (expressed in the fat body, trachea, and a few cells in the gut), en-Gal4 (expressed in posterior epidermal cells, the hindgut, and some neural cells), Act-Gal4 (expressed ubiquitously), and hs-Flp/Act>Cd2>Gal4 (induced by heat shock in all cells). In several cases, overexpressed Dp110 and Inr were tolerated in feeding animals. For instance, animals expressing Dp110 under Adh-Gal4 or en-Gal4 control developed without delay and eclosed at the same frequency as controls, giving viable fertile adults. Inr was more deleterious, but some animals expressing Inr under Adh-Gal4 control developed to the L3 stage and a few viable adults eclosed. Ubiquitous expression of Dp110 or Inr using the Act-Gal4 driver, however, was 100% lethal at prelarval stages (Britton, 2002).

In contrast, hyperactivating Inr/PI3K signaling under starvation conditions was catastrophic. When Adh-Gal4 was used to drive Dp110 or Inr expression, for instance, L1 larvae raised on the sucrose/PBS diet all perished within 3 to 4 days of hatching, whereas control animals survived 8 to 9 days. Animals expressing Dp110 under en-Gal4 control also perished within 2 to 3 days, 4 to 5 days before controls, when deprived of dietary protein. Suppressing PI3K activity by expressing p60, Deltap60, Pten, or Dp110D945A using Adh-Gal4, en-Gal4, or even Act-Gal4 had no effect on viability under starvation conditions. These results suggest that the starvation sensitivity caused by high Inr/PI3K activity is specifically related to nutrient uptake and storage, functions that appear to be unique to Inr and PI3K (Britton, 2002).

These results demonstrate that downregulation of Inr/PI3K activity is critical to maintaining metabolic homeostasis under starvation conditions. The remarkable ability of Inr/PI3K-expressing cells to continue stockpiling nutrients and grow, even in starved animals, may account for the starvation sensitivity observed at the organismal level. This idea was supported by observations made in starved tGPH larvae that overexpressed Dp110 in posterior compartment epidermal cells (genotype, en-Gal4 UAS-Dp110 tGPH). In these animals, high levels of membrane-bound tGPH persisted in Dp110-expressing epidermal cells until 2 days after nutrient withdrawal, at which point the animals died. In anterior (A) cells, which did not overexpress Dp110, tGPH was completely lost from plasma membranes within 18 hr after nutrient deprivation, and tGPH protein became undetectable by 36 hr. This is a much more rapid starvation response than observed in animals that did not contain Dp110-expressing cells. Anterior epidermal cells also shrank rapidly during starvation, whereas posterior, Dp110-expressing cells maintained their large size. These effects might result from the rapid depletion of nutrients by the PI3K-expressing cells, and a consequent drop in levels of hemolymph insulins (Britton, 2002).

In performing these experiments, it was noticed that larvae that overexpress Inr or Dp110 wander away from their food. To more carefully analyze this phenotype, animals expressing various PI3K signaling components under Adh-Gal4 control were cultured on agar plates with red-colored food (yeast paste) in the center for ~24 hr after hatching. These animals were then scored for the presence of red food in the gut as well as their proximity to the food source. Animals overexpressing Inr, Dp110, or Dp110CAAX feed poorly (i.e., often have no food in the gut) and frequently wander away from their food. Similar aberrant behaviors were observed when Dp110 was expressed ubiquitously using Flp/Gal4, in which case nearly all animals wandered out of the food and pupated precociously. Thus, elevated levels of Inr/PI3K signaling alter larval feeding behavior, perhaps by affecting the animal's perceived level of hunger (Britton, 2002).

Is nutrition sensed in Drosophila at the cell level or by the animal as a whole? Although animal cells can sense amino acids directly, these studies suggest that cells in Drosophila larvae sense and respond to changes in dietary protein indirectly, using secondary humoral signals -- most likely insulins -- long before they become acutely starved for amino acids. In support of this idea, some cells in the larva can continue to grow and replicate their DNA long after the animal is deprived of dietary protein. Imaginal cells and neuroblasts do this, as do ERT cells in which Inr or PI3K have been artificially switched on. This attests to the fact that starvation for dietary protein does not completely deplete the larval hemolymph of amino acids or cause a global shutdown of protein synthesis. This is probably possible because nutrients stored in ERTs such as the fat body are mobilized during starvation to maintain levels of hemolymph nutrients. Nevertheless, starvation does cause a rapid, global shutdown of ERT cell growth, and thus some essential signal is lost (Britton, 2002).

One factor that all animal cells use for nutritional sensing is TOR (target of rapamycin), a protein kinase that mediates diverse effects on cell metabolism including protein synthesis, amino acid import, ribosome biogenesis, and autophagy (Raught, 2001; Schmelzle, 2000). What role might Drosophila TOR (dTOR) play in the nutrition response system addressed here? The mechanism by which TOR 'senses' nutrition remains uncertain (Kleijn and Proud, 2000), but cellular levels of amino acids, aminoacylated tRNAs, and ATP have been suggested as direct inputs (Dennis, 2001; Iiboshi, 1999). Drosophila dTOR mutations or the TOR-specific inhibitor rapamycin inactivate the TOR target S6-kinase and phenocopy starvation in fed larvae (Zhang, 2000; Oldham, 2000). Starvation, however, does not completely inactivate S6K, suggesting that dTOR retains some activity under starvation conditions (Oldham, 2000). Consistent with this interpretation, overexpressed PI3K is a potent promotor of cell growth in starved larvae, but PI3K cannot drive cell growth in dTOR mutant larvae (J. Lande and T. Neufeld, personal communication to Britton, 2002). This suggests that although dTOR may act as a cell-autonomous nutrient-dependent checkpoint for metabolism, the larva's physiology is so effective in buffering cells against absolute starvation that this checkpoint is rarely if ever fully engaged. TOR is found in fungi and plants and so seems to be a metabolic regulator that was used prior to the advent of multicellularity. Insulin signaling, which is absent from fungi and plants, probably evolved later when multicellular animals required a system to coordinate and fine-tune metabolism in communities of cells. The insulin system is clearly advantageous, since animals in which it is 'short-circuited' by hyperactivation of Inr or PI3K are unable to tolerate even brief periods of starvation (Britton, 2002).

The most direct evidence that insulin signaling is nutritionally controlled was obtained using a cellular indicator of PI3K activity, tGPH, which is recruited to plasma membranes by the second messenger product of PI3K, PIP3. Subcellular tGPH distributions indicate that PIP3 levels are high in many cell types in fed larvae, but low in larvae that have been starved for protein. Although these changes in PIP3 levels might be due to altered expression of Inr, PI3K, or Pten, expression profiling experiments using cDNA microarrays indicate that levels of p60 and Dp110 mRNA are not depressed in L2 larvae that had been deprived of protein for 4 days. Perhaps the most attractive explanation for the apparent loss of PIP3 upon starvation is that some of the seven Drosophila insulin-like peptides (dILPs) are produced in a nutrition-dependent fashion. Several of the dilp genes are expressed in the larval gut (Brogiolo, 2001) which, as the conduit of nutritional influx, might be expected to mediate metabolic responses to feeding throughout the animal. Other dilps are expressed in the salivary glands, imaginal discs, and small numbers of cells in the central nervous system (Brogiolo, 2001; Britton, 2002).

In mammals, insulin promotes the cellular uptake and storage of carbohydrates, proteins, and lipids, and is the strongest anabolic inducer known. Insulin-mediated responses are indirectly antagonized by the hormone glucagon, which stimulates catabolic reactions and the mobilization of stored nutrients. Insulin and glucagon are produced in the pancreas, and their relative levels are constantly adjusted to maintain proper blood sugar levels. The mammalian liver is also a key player in the regulation of metabolic homeostasis. In humans, most of the accessible glycogen, the principle form of stored carbohydrate, is found in the liver. This glycogen can be mobilized in response to exercise or starvation. In Drosophila larvae, hyperactivation of the Inr/PI3K pathway leads to increased accumulation of nutrients in the fat body, an organ that resembles the mammalian liver as the principal site of stored glycogen. Conversely, inhibition of PI3K activity depletes stored nutrients from the fat body, as does starvation. This suggests that like mammals, insects regulate storage of metabolites in response to changes in levels of Inr/PI3K signaling. Direct assays of the levels of carbohydrates, storage proteins, and lipids in the fat body after starvation or manipulation of Inr/PI3K activity should prove informative. While there are as yet no known Drosophila homologs of glucagons, there must be some mechanism by which stored resources can be mobilized during starvation or at the transition from feeding to metamorphosis (Britton, 2002).

Insulin-like peptides and growth regulation

In the fruit fly Drosophila, four insulin genes are coexpressed in small clusters of cells [insulin-producing cells (IPCs)] in the brain. Ablation of these IPCs causes developmental delay, growth retardation, and elevated carbohydrate levels in larval hemolymph. All of the defects were reversed by ectopic expression of a Drosophila insulin transgene. On the basis of these functional data and the observation that IPCs release insulin into the circulatory system, it is concluded that brain IPCs are the main systemic supply of insulin during larval growth. It is proposed that IPCs and pancreatic islet beta cells are functionally analogous and may have evolved from a common ancestral insulin-producing neuron. Interestingly, the phenotype of flies lacking IPCs includes certain features of diabetes mellitus (Rulifson, 2002).

The Drosophila genome contains five Drosophila insulinlike peptide genes (dilp1 through -5) with significant homology to mouse and human insulins and two others with far less similarity (dilp6 and -7). These genes are expressed in tissues ranging from early embryonic mesoderm to small clusters of larval brain neurons, ventral nerve cord neurons, salivary gland, and midgut. Using messenger RNA (mRNA) in situ hybridization, it has been established that the most prominent insulin gene expression during larval stages, a period of intensive feeding and rapid growth, is within two bilaterally symmetric clusters of neurosecretory cells in the pars intercerebralis region of the protocerebrum (Rulifson, 2002).

An 859-base pair promoter fragment, comprised of sequences immediately 5' of dilp2, is sufficient to drive gene expression in the small clusters of larval brain neurons that express dilp1, -2, -3, and -5. To assess the role of the brain IPCs as an insulin-producing endocrine system, the brain IPCs were ablated using the dilp2 promoter to express the cell death-promoting factor, Reaper. The IPC ablation results in deficiency of brain neuron-derived insulin only. IPC ablation caused undergrowth phenotypes, developmental delays, and lethality similar to Drosophila insulin receptor (DInR) mutants. To rule out an underlying cause of these phenotypes other than insulin deficiency, such as non-IPC death from Reaper or loss of other essential brain IPC functions, a heat shock-inducible dilp2 transgene was used with ubiquitous expression to reverse the effect of the IPC ablation (Rulifson, 2002).

The defect in growth was quantified by comparing larval length after 120 hours of development, a time at which synchronized cultures of normal larvae will reach wandering third instar and puparium formation. After IPC ablation, larvae attained a mean length only 58% of normal size. Larvae with IPCs ablated but expressing the inducible dilp2 transgene had their mean length rescued to 88% of normal. The developmental time to reach wandering third instar and puparium formation was approximately 5 days in normal larvae but lengthened to 12 days in larvae after IPC ablation, a developmental rate similar to that observed in animals homozygous for loss-of-function mutations in DInR. Larvae with ablated IPCs that expressed the inducible dilp2 transgene require approximately 6 days to reach puparium formation. Thus, systemic DILP2 expression is sufficient to compensate for IPC ablation. The fact that brain IPC ablation is rescued by dilp2 alone suggests that insulin made by brain IPCs may be partially redundant (Rulifson, 2002).

IPC ablation produces small-sized adults of normal proportion. Examination of adult wings revealed reductions in both cell size and number after IPC ablation. Under the strongest condition of IPC ablation, mean wing size was reduced to 61% of normal, whereas wing cell number and size were reduced to 72% and 85% of normal, respectively. Under a less severe regimen of IPC ablation, mean wing size was reduced to 74% of normal, with reductions in cell number and size to 81% and 91% of normal, respectively. As in larval growth, the dilp2 transgene effectively reverses the effects of IPC ablation on wing growth and, in fact, caused a slight overgrowth effect, possibly due to the 20% lengthening of developmental time that allowed more growth. The overall reduction in cell size and number after IPC ablation is similar to that in DInR and IRS1-4 mutants. This, together with the observation that brain IPC-derived insulins can activate the DInR in vitro, suggests that brain IPCs are a key source of insulin for this growth control pathway (Rulifson, 2002).

The role of brain IPCs and insulin in the regulation of carbohydrate metabolism was also investigated. Trehalose is a disaccharide composed of two glucose molecules and is the principal blood sugar in many insects. Using the same heat shock regimen, the combined concentration was examined of glucose and trehalose in the hemolymph of wandering third instar larvae, a brief and discrete developmental stage before puparium formation when feeding has ceased. The IPC-ablated larvae had an average combined glucose and trehalose level of 38% above normal, and these levels returned to normal when DILP2 was provided systemically by the transgene. Elevated hemolymph carbohydrate levels in larvae lacking IPCs indicate that insulin is an essential regulator of energy metabolism in Drosophila. This accumulation of carbohydrate in the blood is reminiscent of that seen in human diabetes mellitus, although it should be noted that carbohydrate levels were measured during development rather than in adults (Rulifson, 2002).

To investigate how central nervous system (CNS)-derived insulin regulates systemic functions, the Drosophila IPC contacts outside the CNS were examined. The morphology of the brain IPCs was examined with the use of the dilp2 promoter to drive expression of mGFP, a membrane bound green fluorescent protein (GFP). The IPC clusters within the pars intercerebralis extend processes that terminate at the lateral protocerebrum and subesophageal ganglion. IPC processes also terminate on the heart and in the corpora cardiaca (CC) compartment of the ring gland, after crossing the midline and exiting the CNS. Labeling of the IPC processes with mGFP and DILP2 antibody revealed localization of DILP2 peptide within the processes that contact the heart and ring gland. DILP2 peptide is concentrated in a graded distribution outside the cells of synthesis on the heart, and colabeling with myosin heavy chain antibody, which labels the columnar heart epithelium, showed that the IPC processes and DILP2 are localized outside the lumen of the heart. These results suggest the heart surface may be the site of insulin release to the openly circulating hemolymph. It is proposed that brain IPCs are essential for organism-wide growth control and carbohydrate homeostasis through release of insulin peptides into circulating hemolymph. These cellular functions are notably similar to those of mammalian pancreatic ß cells (Rulifson, 2002).

In Drosophila and other insects, a fraction of CC cells synthesize adipokinetic hormone (AKH). AKH resembles glucagon in its activation of glycogen phosphorylase through heteromeric GTP-binding protein (G protein) and adenosine 3',5'-monophosphate (cAMP) signaling to elevate blood sugar, and the two proteins have some limited sequence similarity. Double labeling of AKH mRNA and DILP2 peptide shows that IPCs extend processes to the CC and that AKH-expressing cells contain DILP2. CC cells accumulate DILP2 within membrane-bound particles of the perinuclear space, suggesting the possibility that DILP2 is taken up by AKH cells. The possibility that dilp2 is transiently or minutely transcribed by AKH cells cannot be ruled out, although expression of either the dilp2 promoter or dilp1, -2, -3, and -5 mRNAs in the AKH cells has not been detected. Thus, in addition to contacts between IPCs themselves, the primary sites of IPC contact outside the CNS are the heart and the CC. Though they lack strict morphological homology, these intercellular contacts are analogous to the association of pancreatic ß cells with other ß cells, with glucagon-expressing a cells, and with blood vessels in the islets of Langerhans and may reflect underlying evolutionary conservation (Rulifson, 2002).

Thus, there is remarkable similarity of the organ systems underlying conserved insulin function in diptera and mammals. Moreover, the presence of IPCs in the nervous systems of other invertebrate and protochordate species and in primary cell cultures from mammalian fetal brain provides further evidence for a common ancestral insulin-producing organ of neural origin. These results also raise the possibility that common mechanisms of cell specification regulate development of pancreatic a cells and Drosophila brain IPCs (Rulifson, 2002).

Nutrient-dependent expression of Insulin-like Peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila

The insulin/IGF-1 signaling pathway controls cellular and organismal growth in many multicellular organisms. In Drosophila, genetic defects in components of the insulin signaling pathway produce small flies that are delayed in development and possess fewer and smaller cells as well as female sterility, reminiscent of the phenotypes of starved flies. This study establishes a causal link between nutrient availability and insulin-dependent growth. In addition to the Drosophila insulin-like peptide 2 (dilp2) gene, overexpression of dilp1 and dilp3-7 is sufficient to promote growth. Three of the dilp genes are expressed in seven median neurosecretory cells (m-NSCs) in the brain. These m-NSCs possess axon terminals in the larval endocrine gland and on the aorta, from which DILPs may be released into the circulatory system. Although expressed in the same cells, the expression of the three genes is controlled by unrelated cis-regulatory elements. The expression of two of the three genes is regulated by nutrient availability. Genetic ablation of these neurosecretory cells mimics the phenotype of starved or insulin signaling mutant flies. These results point to a conserved role of the neuroendocrine axis in growth control in multicellular organisms (Ikeya, 2002).

The insulin/IGF signaling pathway plays a key role in the control of growth in vertebrates and invertebrates. In mammals, the primary role of insulin and the insulin receptor is energy homeostasis by the regulation of blood glucose levels. But mutations in the human insulin receptor gene also cause embryonic growth retardation. The primary growth regulatory function in mammals, however, is mediated by the IGF-1 and IGF-2 growth factors and the IGF-1 receptor. In Drosophila, there is a single insulin-like receptor, and it regulates postembryonic growth, reproduction, and aging. In vertebrates and invertebrates, intracellular signal transduction from the insulin/IGF receptors depends on insulin receptor substrate (IRS) proteins. In mammals, loss of IRS1 function causes severe reduction in embryonic and postembryonic growth (Araki, 1994; Tamemoto, 1994). Loss of IRS2 leads only to a moderate reduction in growth, but mice become hyperglycemic and contain increased body fat, and females are sterile (Withers, 1998). In Drosophila, flies mutant for chico, which encodes the single Drosophila homolog of IRS1-4, are developmentally delayed, have a severely reduced body size and increased fat, and females are sterile. This demonstrates a striking conservation of the role of the insulin/IGF signaling pathway during evolution (Ikeya, 2002 and references therein).

Several lines of evidence suggest a link between the activity of the insulin/IGF signaling pathway and nutrient availability. The female sterility and the growth retardation phenotypes of IRS2-/- and IRS1-/- mice, respectively, are similar to those observed in starved mice (Butler, 2001, Tiessen, 1999). In Drosophila, growth retardation, reduced body weight due to fewer and smaller cells, and female sterility are phenotypes not only characteristic of chico mutant flies but also of flies that have been starved during development. In fact, oogenesis is blocked during stem cell divisions and before the onset of vitellogenesis in starved flies and in chico mutant flies. Furthermore, starvation reduces the activity of the insulin signaling pathway in vivo. In nematodes, starvation or mutations in the insulin signaling pathway arrest development at the so-called Dauer stage. Moreover, caloric restriction and mutations in the insulin/IGF1 signaling system extend life span in vertebrates and invertebrates. It is not known, however, whether the link between nutrient availability and the activity of the insulin signaling pathway is direct, and how the nutritional status is translated into insulin/IGF receptor activity in the target tissues (Ikeya, 2002 and references therein).

One obvious hypothesis for a connection between nutrient availability and insulin/IGF activity is that nutrients control the expression of the insulin/IGF growth factors. In mammals, blood glucose levels not only regulate the release of insulin from the pancreatic ß cells, but also regulate the insulin gene expression via an autocrine loop (Xu, 2001). The regulation of IGF-1 levels that control postnatal growth is more complex. Growth hormone synthesized by the pituitary controls IGF-1 expression in the liver, accounting for approximately one-third of the postnatal growth promoting activity. Growth also depends on GH-independent expression of IGF-1 and GH activity that is independent of IGF-1 (Ikeya, 2002 and references therein).

The role of insulin-like growth factors in invertebrates in the regulation of growth and their regulation in response to starvation is less well defined. In Drosophila, the search for insulin-like genes (dilps) in the genome has revealed seven genes that show highly regulated temporal and spatial expression. Ubiquitous overexpression of one of the dilp genes, dilp2, is sufficient to increase body size. This growth-promoting activity of DILP2 is dependent on the insulin receptor signaling pathway. Three dilp genes are expressed in small cell clusters in the central region of the brain. These three genes are expressed in the same median neurosecretory cells (m-NSCs) possessing axon terminals in the ring gland and the corpora cardiaca. Although all three peptides can promote growth when overexpressed, they are regulated differentially in response to starvation. Furthermore, genetic ablation of the m-NSCs produces a phenotype reminiscent of chico mutant flies (Ikeya, 2002).

Expression of dilp2, 3, and 5 genes is detected in the same two clusters of cells. The position of these cell clusters in the pars intercerebralis of the larval brain suggests that they correspond to the m-NSC clusters that stain with an anti-Bombyxin antibody. Indeed, the expression of GFP under the control of the dilp2 cis-regulatory elements permits the visualization of the axonal projections of the dilp expressing cells. Axon projections are seen in the corpora cardiaca of the ring gland and on the aorta, the site from which neuropeptides are released into the hemolymph. Therefore, dilp2, 3, and 5 are coexpressed in the cluster of m-NSCs identified in larger insects and in Drosophila. Although expressed in the same cells, the temporal expression pattern of dilp 2, 3, and 5 in the m-NSCs differs. While dilp2 is expressed already in the first instar stage, dilp2 and dilp5 expression is detectable in the second instar stage, while dilp3 expression starts at the mid to late third instar stage. The successive activation of dilp genes in the m-NSCs correlates with the increasing growth that occurs during the last larval instar (Ikeya, 2002).

The dilp2, 3, and 5 genes are located together with dilp1 and dilp4 in a gene cluster spanning 26 kb on the third chromosome. To search for enhancer elements controlling the expression of the three genes in the m-NSCs, a series of lacZ reporter genes containing various amounts of upstream genomic sequences was constructed. Upstream fragments of 1.0 kb, 1.7 kb, and 450 bp derived from the dilp2, 3, and 5 genes, respectively, are sufficient to drive reporter gene expression specifically in the m-NSC. Thus, each of the three genes possesses its own m-NSC-specific enhancer. A further dissection of these upstream sequences has revealed that a 394 bp fragment located at position -540 to -146 of dilp2 is sufficient to recapitulate the expression of dilp2 in the m-NSCs. This construct, however, is no longer expressed in imaginal discs, suggesting that different enhancer elements control expression in this tissue. The cis-regulatory elements that control dilp3 expression are more complex. The m-NSC-specific expression depends on two separate elements located between -763 to -1167 and between +1 to -165, including the putative transcription start site. The fragment located between the m-NSC enhancers drives expression of dilp3 in gut muscles (Ikeya, 2002).

Surprisingly, sequence comparison of the genomic sequences required for m-NSC expression of dilp2, 3, and 5 did not reveal obvious stretches of similarity that may identify a common m-NSC-specific enhancer element in the different genes. It therefore appears that even within this small group of cells, the three dilp genes are regulated by different combinations of transcription factors (Ikeya, 2002).

A causal link between starvation-induced reduction in growth and reduced insulin receptor activity may involve the regulation of circulating DILP levels by nutrient availability. To test this hypothesis, the expression of dilp2, 3, and 5 was examined in third instar larvae that had been starved for 24 hr. In nonstarved larvae, mRNA transcripts of dilp2, 3 and dilp5 were detected in the m-NSCs. Upon starvation, dilp3 and dilp5 transcript levels are reduced, while dilp2 transcript levels remain unchanged. Similar results were obtained when the dilp5-lacZ reporter construct was used. In these larvae, lacZ mRNA is severely reduced upon starvation, while LacZ activity is still detectable owing to the stability of the LacZ protein. Therefore, the enhancer elements that respond to starvation are located in the 450 bp fragment used to analyze dilp5 expression. These results demonstrate that at least part of the nutrient-dependent regulation of growth is mediated by the regulated expression of the dilp3 and dilp5 genes (Ikeya, 2002).

During larval development, the seven dilp genes are expressed in a variety of different tissues in addition to the m-NSCs. To begin to address the role of DILP production in the m-NSCs, these cells were specifically ablated. The dilp2-Gal4 line, which is exclusively expressed in the m-NSCs starting at the late third instar stage, was used to drive expression of the proapoptotic gene reaper (rpr) in these cells. dilp2:rpr flies are viable but eclose one day later than control flies. Freshly eclosed flies show a slight but significant reduction in body weight. The size difference is also observed in the reduced wing area. Interestingly, the largest difference between control and dilp2:rpr flies is observed in the size of the abdomen of females. This difference becomes further enhanced during the first three days of adult life. During this phase, egg production is stimulated by feeding and mating in wild-type females. Comparison of the ovaries of three-day-old wild-type and dilp2:rpr females revealed a striking difference in ovary size. Each ovary is composed of approximately 15 ovarioles. Ovarioles are oocyte tubes containing stem cells at the tip and oocytes of increasing maturity toward the oviduct. While wild-type females possess multiple vitellogenic oocytes in each ovariole, dilp2:rpr females possess at most a single vitellogenic oocyte. This reduced size of the ovary of dilp2:rpr flies is reflected in the reduced fecundity of these females. While control flies lay on average 60 eggs per day, dilp2:rpr females produce only 10 eggs per day. The dilp2:rpr flies exhibit a developmental delay, reduced body size, and reduced fecundity of females owing to a partial block in production of vitellogenic oocytes. These phenotypes resemble those of weak mutations in the genes coding for components of the insulin signaling pathway. While chico females are almost completely sterile and severely reduced in size, certain combinations of Inr alleles produce females very similar to the dilp2:rpr flies. It is possible that the partially penetrant phenotype of m-NSC ablation is due to the late onset of expression of the dilp2-Gal4 driver line, resulting in only a partial ablation of the m-NSCs. Since dilp5 expression is also observed in the follicle cells of the female ovary, this m-NSC independent source of DILP may provide an alternative explanation for the partially penetrant phenotype of m-NSC ablation (Ikeya, 2002).

Nutrient-dependent regulation of growth and reproduction is observed in all multicellular organisms. The results presented here provide further support for an evolutionary conserved signaling pathway involved in this regulation. In mammals, insulin secretion from the pancreatic ß cells is regulated by the concentration of glucose in the blood, and thereby regulates energy homeostasis in response to food intake. Embryonic and postembryonic growth is regulated by IGF-1 production that in part depends on GH synthesized from the pituitary. In insects, the m-NSCs appear to play a role in both functions. The release of insulin-like peptides from the corpora cardiaca in Bombyx is regulated by carbohydrate levels in the hemolymph (Masumura, 1997) in a way analogous to the release of glucose from pancreatic ß cells in mammals. Expression of dilp3 and dilp5 is repressed by food withdrawal. Furthermore, ablation of the m-NSCs results in growth retardation. These results are consistent with a recent study showing that early Reaper-induced ablation of the m-NSC cells using a multimerized dilp2-Gal4 construct severely reduced growth and led to a concomitant increase in glucose and trehalose levels in the hemolymph of these animals (Rulifson, 2002). Given the data from Bombyx and Drosophila together, it is suggested that the m-NSCs possess functions similar to those of the pancreas and the pituitary in mammals (Ikeya, 2002).

In insects, the growth regulatory function of DILPs is 2-fold. (1) Circulating levels of DILPs in the hemolymph activate growth in the target tissues by the activation of the insulin receptor PI3K pathway in each cell. This action is complemented by the local production of DILPs in the target tissues in a manner similar to the expression of IGF-1 in target tissues. (2) DILPs exert their effect on growth indirectly. The m-NSCs project their axon terminals into the ring gland where ecdysone and JH are synthesized. The stimulation of ecdysone synthesis by insulin-like peptides is well documented in many insects. Furthermore, JH levels are reduced in long-lived insulin receptor mutant flies, suggesting that DILPs also regulate JH synthesis. Through the regulation of the levels of one or both of these two hormones, DILPs may regulate growth indirectly. Under starvation conditions, reduced JH levels may result in the premature increase in ecdysone titer in third instar larvae, leading to the precocious initiation of metamorphosis and thus producing flies with fewer cells. Alternatively, a precocious rise in ecdysone titer may be caused by the increase in the local concentration of DILPs in the ring gland due to the increased retention of insulin-like peptides in the corpora cardiaca during starvation (Ikeya, 2002).

In nematodes, nutrient availability regulates the developmental program and fertility without having a direct effect on cell size or cell number. In the absence of food, the larvae enter the immature long-lived Dauer stage. This response is controlled by two pathways, the insulin signaling pathway and the daf-4/TGF-ß pathway. Each of these pathways acts nonautonomously in the nervous system, and they converge on the nuclear hormone receptor daf-12. This implies an intermediate steroid or lipid hormone signal. Indeed, daf-9 that acts genetically between daf-2 and daf-4 signaling encodes a cytochrome P450 enzyme involved in steroid and fatty acid metabolism. It is interesting to note that the two activities of the Prothoracicotropic hormone (PTTH) that regulate ecdysone synthesis in Bombyx involve a member of the TGF-ß superfamily and an insulin-related peptide synthesized in distinct sets of neurosecretory cells in the brain. Therefore, it is likely that the nutrient-dependent growth regulation in nematodes and Drosophila is conserved in spite of the absence of an autonomous requirement of insulin signaling in cell growth in C. elegans (Ikeya, 2002).

Egg maturation is blocked by starvation in many species. In insects, ecdysone produced by the ovary is required for yolk protein production in the fat body and oocyte maturation. Ecdysone production is stimulated by insulin-like peptides in vitro and in vivo. Ablation of m-NSCs significantly slows down oogenesis. In humans, brain-specific knockouts of the insulin receptor or IRS2 also block oocyte maturation by affecting the synthesis of gonadotropins. Furthermore, steroidogenesis in the female gonad is required for oocyte maturation and is regulated by the expression of IGF-1 in different gonadal cells. Interestingly, expression of dilp5 is also detected in the ovarian follicle cells in Drosophila. Local production of DILP5 may stimulate ecdysone production in the female ovary directly. The similar roles of insulin-related peptides in growth regulation, energy homeostasis, and oogenesis in nematodes, insects, and mammals are striking. How far the underlying mechanisms are also conserved remains to be investigated (Ikeya, 2002).

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

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

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

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

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

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

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

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

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

Transcriptional feedback control of insulin receptor by dFOXO/FOXO1

The insulin signaling pathway evolved to allow a fast response to changes in nutrient availability while keeping glucose concentration constant in serum. This study shows that, both in Drosophila and mammals, insulin receptor (InR) represses its own synthesis by a feedback mechanism directed by the transcription factor dFOXO/FOXO1. In Drosophila, dFOXO is responsible for activating transcription of dInR, and nutritional conditions can modulate this effect. Starvation up-regulates mRNA of dInR in wild-type but not dFOXO-deficient flies. Importantly, FOXO1 acts in mammalian cells like its Drosophila counterpart, up-regulating the InR mRNA level upon fasting. Mammalian cells up-regulate the InR mRNA in the absence of serum, conditions that induce the dephosphorylation and activation of FOXO1. Interestingly, insulin is able to reverse this effect. Therefore, dFOXO/FOXO1 acts as an insulin sensor to activate insulin signaling, allowing a fast response to the hormone after each meal. These results reveal a key feedback control mechanism for dFOXO/FOXO1 in regulating metabolism and insulin signaling (Puig, 2005).

It is well known that the expression and activity of the InR can be regulated by a wide variety of factors and that changes in the numbers of receptor molecules plays a pivotal role in several physiologic and pathologic states. The lowered sensitivity of cells to insulin and the hyperinsulinemia observed in obesity and type II diabetes mellitus is often accompanied by a reduced number of insulin receptors. Insulin is thought to down-regulate its own receptor by a variety of mechanisms that can influence its synthesis as well as degradation. Interestingly, it has been shown that the number of InR molecules correlates with nutritional conditions both in tissue culture cells and in animals. Thus, levels of InR in growing HepG2 cells are relatively low, and they increase substantially if cells are starved. In addition, states of chronic hyperinsulinemia produce a reduction in the number of InR present in the plasma membrane. InR mRNA levels also change in animals depending on fasting-feeding conditions. For example, rats fed a high-fat diet display a decreased number of InR molecules in liver plasma membranes, and InR mRNA levels in rat skeletal muscle or liver increase after fasting, returning to normal levels after insulin treatment or refeeding. Interestingly, tissues other than muscle or liver might have similar regulation. For example, mRNA and protein levels of rat intestinal InR increase up to 230% in fasting conditions, and these effects are fully reversed by refeeding. Similar observations have been made in other organisms. These effects indicate a nutritional influence on the abundance of the InR. Importantly, insulin levels in serum change in parallel to nutrient availability, both in flies and mammals. Thus, when nutrients are high—that is, after a meal—insulin levels increase, while they decrease upon fasting. In Drosophila it has been shown that the InR/PI3K pathway coordinates cellular metabolism with nutritional conditions. Inhibiting this pathway phenocopies the cellular and organismal effects of starvation, while activating it bypasses the nutritional requirements for cell growth. The InR/PI3K pathway regulates the activity of FOXO1 in mammals, C. elegans, and Drosophila, so nutrient activation of the PI3K pathway results in inactivation of FOXO1 by phosphorylation. However, despite this accumulated base of information, the molecular mechanism linking FOXO1 and InR expression had not been revealed (Puig, 2005).

This study shows that mammalian FOXO1 and its Drosophila counterpart dFOXO directly regulate insulin-signaling response to nutritional conditions through a feedback mechanism that involves activation of transcription from the InR promoter. Incubating C2C12 cells with a balanced salt solution or with serum-free medium up-regulates insulin receptor mRNA. Under these conditions, FOXO1 becomes dephosphorylated and actively binds to the InR promoter. When insulin is added to the medium, InR mRNA is down-regulated, even in the absence of serum, vitamins, amino acids, and glucose. Concomitantly, phosphorylation of FOXO1 increases and binding to InR promoter decreases. These results indicate that FOXO1 regulates InR transcription through a direct feedback mechanism that senses insulin levels in serum, which is, in turn, a reflection of nutrient load. It is important to note that, at this point, it cannot be ruled out that the increased InR protein levels caused by FOXO1 could be due to other mechanisms in addition to increased transcription from the InR promoter (i.e., affecting mRNA stability, or protein translation) (Puig, 2005).

In Drosophila a similar mechanism occurs. Incubation of S2 cells with complete medium keeps dFOXO phosphorylated and inactive, while incubation in HBSS dephosphorylates dFOXO. dInR mRNA is up-regulated only when dFOXO is dephosphorylated and active. In addition, wild-type flies starved for 4 d up-regulate dInR, and this effect requires an intact dfoxo gene. These studies indicate that in Drosophila, the PI3K/Akt pathway also senses insulin levels and regulates binding of dFOXO to the dInR promoter accordingly. These results underscore the importance of the InR/PI3K/Akt pathway in sensing nutrients and insulin, a function that has been conserved during evolution. They also highlight the role of FOXO1 as a sensor for insulin levels, promoting accumulation of InR in the absence of insulin, thereby allowing a fast response to the hormone after each meal. Under conditions in which insulin levels are chronically elevated, for example, in obese animals or patients, down-regulation of InR transcription would occur and insulin sensitivity would be impaired. These results establish the FOXO1 transcription factor as a key player in a feedback control mechanism that regulates metabolism and insulin signaling (Puig, 2005).

The results show that in conditions in which insulin levels are low, mammalian FOXO1 activates InR. Interestingly, it was observed that FOXO1 also activates the insulin receptor substrate-2 (IRS-2) promoter under fasting conditions, and, since it occurs with InR, insulin is sufficient to reverse this effect. FOXO1 binds IRS-2 promoter in vitro and in vivo and activates IRS-2 transcription when muscle or liver cells are fasted. In addition, FOXO1 activates IRS-2 promoter in luciferase assays, and this activation depends on the presence of a consensus FRE present in the IRS-2 promoter, because mutating this FRE abolishes FOXO1-dependent activation. Thus, FOXO1 regulation of IRS-2 is parallel to InR regulation. It has also been reported that SREBPs compete with FOXO transcription factors for binding to the IRS-2 promoter in liver; while SREBPs inhibit IRS-2 production, FOXO1 was found to activate IRS-2 transcription. It was also found that fasting promotes binding of FOXO1 to the FRE of the IRS-2 promoter. Therefore, these findings strongly support the conclusions that FOXO1 regulates insulin signaling through a feedback mechanism that impinges on the insulin receptor and at least one of its substrates, IRS-2. After a meal, high levels of insulin peptide hormone activate its cognate receptor, which leads to repression of InR and IRS-2 transcription, resulting in subsequent dampening of the pathway by reducing the number of receptors on the cell surface and by limiting its ability to signal downstream through IRS-2. Conversely, fasting causes reduced levels of InR signaling, which in turn activates FOXO1, leading to increased transcription of InR and IRS-2. Once this transcription mechanism is activated, feedback regulation and phosphorylation of FOXO1 via the insulin signaling cascade automodulates InR expression. Insulin sensitivity could, therefore, be significantly affected by FOXO1 regulation. Regulation of insulin sensitivity by a feedback loop through FOXO1 would allow the cells to keep an exquisite metabolic balance between feeding and fasting states, permitting a faster response of the tissues to insulin changes. This feedback mechanism could well be disrupted in pathological states with abnormally increased insulin levels as is found in the case of insulin-resistant diabetes (Puig, 2005).

Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila: Potential role of insulin pathway

Hunger elicits diverse, yet coordinated, adaptive responses across species, but the underlying signaling mechanism remains poorly understood. This study reports on the function and mechanism of the Drosophila insulin-like system in the central regulation of different hunger-driven behaviors. Overexpression of Drosophila insulin-like peptides (DILPs) in the nervous system of fasted larvae suppresses the hunger-driven increase of ingestion rate and intake of nonpreferred foods (e.g., a less accessible solid food). Moreover, up-regulation of Drosophila p70/S6 kinase activity in DILP neurons leads to attenuated hunger response by fasted larvae, whereas its down-regulation triggered fed larvae to display motivated foraging and feeding. Finally, evidence is provided that neural regulation of food preference but not ingestion rate may involve direct signaling by DILPs to neurons expressing neuropeptide F receptor 1, a receptor for neuropeptide Y-like neuropeptide F. This study reveals a prominent role of neural Drosophila p70/S6 kinase in the modulation of hunger response by insulin-like and neuropeptide Y-like signaling pathways (Wu, 2006).

The relatively simple Drosophila larva offers a genetically tractable model to define and characterize different neuronal signaling pathways that constitute a complete central feeding apparatus. Younger third-instar larvae forage actively and use their mouth hooks for food intake. Larvae normally feed on liquid food, and their food ingestion can be quantified by measuring the contraction rate of the mouth hooks. This study examined how food deprivation affects larval feeding response to a liquid (e.g., 10% glucose-agar paste) and less accessible solid food (e.g., 10% glucose agar blocks). To extract embedded glucose from the solid food, larvae have to pulverize the food by scraping agar surface with mouth hooks. Unless stated otherwise, synchronized third-instar larvae (74 h after egg laying) were used for the assays (Wu, 2006).

When fed ad libitum, normal larvae (w1118) display significant feeding activity in the liquid food with an average mouth-hook contraction frequency of ~30 times in a 30-s test period; in contrast, these larvae declined the solid food. However, larvae withheld from food (on a wet tissue) for 40 or 120 min display increased intake of both liquid and solid foods. For example, larvae fasted for 120 min show a 100% and >500% increase in mouth-hook contraction rate in liquid and solid food, respectively. Thus, deprivation not only enhances feeding rate in a graded fashion, but also triggers motivated foraging on the less accessible food normally rejected by fed larvae. In addition, larvae display virtually identical feeding responses to liquid and solid foods containing 10% glucose, apple juice, or 10% glucose/yeast under deprived and nondeprived conditions. Therefore, these paradigms appear to provide a general assessment of larval feeding response (Wu, 2006).

dS6K is a cell-autonomous effector of nutrient-sensing pathways. This study investigated a possible role of neural dS6K in coupling peripheral physiological hunger signals and neuronal activities critical for hunger-driven behaviors. The transcripts of dilp1, dilp2, dilp3, and dilp5 are predominantly expressed in two small clusters of medial neurosecretory cells that project to the ring gland, the fly heart, and the brain lobes. A gal4 driver containing a 2-kb fragment from the dilp2 promoter (dilp2-gal4) was generated that directs the specific expression of a GFP reporter in those cells. Using dilp2-gal4, two transgenes, UAS-dS6KDN, encoding a dominant negative, and UAS-dS6KACT, a constitutively active form of dS6K, were expressed. When fed ad libitum, control larvae (w x UAS-dS6KDN or UAS-dS6KACT) behave like w larvae. However, dilp2-gal4 x UAS-dS6KDN larvae displayed a 50% increase in the rate of liquid-food intake and significant feeding of the solid food. Conversely, fasted larvae overexpressing dS6K activity (dilp2-gal4 x UAS-dS6KACT) showed attenuated feeding response to both liquid and solid foods. These findings reveal that dS6K in DILP neurons mediates hunger regulation of approaching/consumptive behaviors, controlling both quality and quantity of food for ingestion. The body size and the developmental rate of all four groups of larvae were measured, and no significant differences were detected (Wu, 2006).

DILPs act as neurohormones in Drosophila larvae. Down-regulation of dS6K activity in DILP neurons may reduce DILP release, thereby promoting increased food intake that is normally triggered only by hunger. A corollary of this interpretation is that overproduction of DILPs in the nervous system should interfere with hunger response by deprived animals. To test this idea, a neural-specific elav-gal4 driver was used to direct dilp expression in the larval nervous system. Three UAS-dilp lines (UAS-dilp2, UAS-dilp3, and UAS-dilp4) were chosen for the analysis. The elav-gal4 x UAS-dilp2 and UAS-dilp4 larvae displayed normal feeding response when fed ad libitum. However, the same larvae fasted for 120 min displayed significantly attenuated feeding rates, similar to those of dilp2-gal4 x UAS-dS6KACT larvae. For example, the comparative analysis of the elav-gal4 x UAS-GFP control and elav-gal4 x UAS-dilp2 and UAS-dilp4 experimental larvae showed that the latter were ~30% and 33–45% lower in the ingestion rate of the liquid and solid food, respectively; surprisingly, elav-gal4 x UAS-dilp3 and UAS-GFP larvae showed virtually identical feeding responses. Therefore, DILP2 and DILP4 negatively regulate hunger-driven feeding activities. Taken together, these results suggest that a high level of dS6K activity in DILP neurons may suppress hunger response by reducing DILP release (Wu, 2006).

Attempts were made to delineate the signaling mechanism that couples the dS6K activity in DILP neurons with its broad impact on hunger-driven feeding activities. A previous study showed that fasted larvae ablated of NPF or its receptor (NPFR1) neurons are deficient in motivated feeding of the less-preferred solid food but normal in feeding of richer liquid food. It was of interest to enquire whether the NPF/NPFR1 neuronal pathway might be one of the downstream effectors of the DILP pathway. To test this hypothesis, the function of three components of the dInR signaling pathway were analyzed in NPFR1 neurons: dInR, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase (dPTEN), and phosphatidylinositol 3-kinase (dPI3K). Five different transgenes were used: UAS-dInRACT and UAS-dInRDN encode a constitutively active and a dominant-negative form of dInR, respectively; UAS-Dp110 and UAS-dPI3KDN encode a catalytic subunit and a dominant-negative form of dPI3K, respectively; and UAS-dPTEN encodes a functional enzyme. When fed ad libitum, npfr1-gal4 x UAS-dInRDN, UAS-dPTEN, or UAS-dPI3KDN larvae display hyperactive feeding of the solid food, similar to w larvae deprived for 40 min. In contrast, fasted larvae overexpressing dInR or dPI3K (npfr1-gal4 x UAS-dInRACT or UAS-Dp110) display attenuated feeding response to the solid food. Importantly, larvae with up- or down-regulated dInR signaling in NPFR1 neurons do not exhibit significant changes in the intake rate of the richer liquid food relative to the paired controls. Taken together, these findings suggest that the dInR pathway negatively regulates the activity of NPFR1 neuron and mediates the DILP-regulated change in food preference but not ingestion rate. Furthermore, the results suggest that NPFR1 neurons are the direct targets of DILPs (Wu, 2006).

A possible role of dS6K in hunger regulation of the functioning of NPFR1 neurons was evaluated, by expressing UAS-dS6KDN and UAS-dS6KACT using npfr1-gal4. When fed ad libitum, npfr1-gal4 x UAS-dS6KDN larvae display hyperactive feeding of the solid food, similar to npfr1-gal4 x UAS-dInRDN larvae. However, these larvae, unlike dilp2-gal4 x UAS-dS6KDN animals, display no increases in the ingestion rate of the richer liquid food. Conversely, fasted larvae overexpressing dS6K (npfr1-gal4 x UAS-dS6KACT) display attenuated feeding response to the solid food. These findings suggest that dS6K also negatively regulates the activity of NPFR1 neurons in food preference, but does not mediate the regulation of feeding rate by DILP signaling (Wu, 2006).

The food response was evaluated of the solid and liquid food by larvae overexpressing an npfr1 cDNA under the control of an npfr1-gal4 driver. In the presence of the liquid food, both experimental (npfr1-gal4 x UAS-npfr1) and control larvae (e.g., npfr1-gal4 x UAS-ANF-GFP), fed or fasted, show similar intake rates and comparable increases in feeding response to hunger. However, when forced to feed on the solid food, fed experimental larvae exhibit significant intake of the solid food (30 times per 30 s), whereas fed controls rejected the same food. Thus, NPFR1 overexpression selectively promotes change in food preference without increasing ingestion rate. It was also observed that the feeding responses of NPFR1-overexpressing larvae and controls fasted for 120 min were indistinguishable. Thus, the effect of NPFR1 overexpression on food preference is detectable only in fed or mildly fasted larvae, suggesting hunger-activated NPFR1 signaling approaches a plateau in severely fasted animals (Wu, 2006).

npfr1 activity was selectively knocked down by expressing npfr1 dsRNA in the nervous system. The UAS-npfr1dsRNA lines were previously used to functionally disrupt npfr1 activity. It was found that 120-min fasted larvae expressing npfr1 dsRNA in NPFR1 or the nervous system (npfr1-gal4, elav-gal4, or appl-gal4 x UAS-npfr1dsRNA) were deficient in motivated feeding of the solid but not liquid food. In contrast, all control larvae, including those expressing npfr1dsRNA in muscle cells (MHC82-gal4 x UAS-npfr1dsRNA), showed normal feeding responses. These results indicate that neural NPFR1 mediates hunger regulation of food selection (Wu, 2006).

A potential problem of the previous transgenic studies is that NPF/NPFR1 signaling is likely to be disrupted in a relatively early stage of larval development. Conceivably, the NPF/NPFR1 neuronal pathway could be essential for ad libitum or hunger-driven feeding of richer liquid foods, but such an activity might be masked by some yet-unidentified compensatory mechanism triggered by its early loss. To test this idea, attempts were made to disrupt NPF/NPFR1 neuronal signaling in a temporally controlled manner by expressing a temperature-sensitive allele of shibire (shits1) driven by npf-gal4 or npfr1-gal4. The shits1 allele encodes a semidominant-negative form of dynamin that blocks neurotransmitter release at a restrictive temperature (>29°C). At the permissive temperature of 23°C, 120-min-fasted experimental larvae (npf-gal4 and npfr1-gal4 x UAS-shits1) and paired controls (y w x UAS-shits1 and npf-gal4 and npfr1-gal4 x w1118) displayed normal feeding responses to both liquid and solid foods. However, if larvae were incubated at 30°C for 15 min, controls still displayed normal feeding activities, whereas the experimental larvae showed attenuated feeding response to the solid but not liquid food. Therefore, there was no detectable developmental or physiological compensation for the loss of NPF signaling in Drosophila larvae. These results also suggest that the NPF/NPFR1 neuronal pathway is acutely required to initiate and maintain larval hunger response. The foraging activity of the experimental larvae was completely restored when the assay temperature was reduced to 23°C, suggesting that the NPF system can modulate the intensity and duration of feeding response (Wu, 2006).

This study has shown that dS6K regulates different, yet coordinated, behaviors controlling quantitative and qualitative aspects of hunger-adaptive food response. Evidence is provided that dS6K mediates hunger regulation of two opposing insulin- and NPY-like signaling activities, dynamically modifying larval food preference and feeding rate based on the nutritional state. For example, hunger stimuli may cause a reduction of dS6K activity in DILP neurons, resulting in the suppression of DILP signaling that negatively regulates a downstream NPF/NPFR1-dependent and another NPF-independent neuronal pathway. The DILP/NPFR1 neuronal pathway selectively mediates hunger-adaptive change in food preference, possibly by overriding the high threshold of food acceptance set by a separate default pathway, enabling hungry animals to be receptive to less preferred foods. The NPF/NPFR1-independent pathway promotes a general increase in the ingestion rate of preferred/less preferred foods, enabling animals to compete effectively for limited food sources. This study also implicates the presence of a separate default pathway for mediating the selective intake of preferred foods (baseline feeding) in larvae fed ad libitum. This default pathway may be largely insensitive to DILP or NPF signaling, because overexpression of dS6K, DILPs, or NPFR1 in nondeprived larvae does not affect ad libitum feeding in the liquid food. It is suggested that the conserved S6K pathway may be critical for regulating behavioral adaptation to hunger in diverse organisms, including humans, and its components are potential drug targets for appetite control (Wu, 2006).

The functional differences of DILP1–7 have not been reported previously. In this study, dilp2, dilp3, and dilp4 were shown to be functionally distinct. DILP2 and DILP3 both are produced in the same medial neurosecretory cells. However, unlike DILP2, DILP3 is apparently not involved in suppressing deprivation-motivated feeding. It is still unclear whether the differential activities of DILP2 and DILP3 reflect their structural divergence or are caused by the presence of yet-unidentified dInR isoforms. DILP4 is not expressed within the two medial clusters of DILP neurons. Under acute deprivation, the level of dilp4 transcripts showed a 5-fold reduction in the larval CNS. Thus, it is possible that DILP4 may play a localized role in promoting feeding response inside the CNS (Wu, 2006).

Feeding is a reward-seeking behavior, and deprivation strengthens the reinforcing effect (reward value) of food. These studies suggest a previously uncharacterized role of the DILP/dInR signaling pathway in regulating an animal's perception of food quality. The DILP/NPF neural network may regulate an animal's incentive to acquire lower-quality foods by modifying the reward circuit. This hypothesis is interesting in light of the findings that foods and abused substances may act on the same reward circuit, and highly palatable foods can reduce drug-seeking behaviors. It is also possible that the DILP/NPF system might represent a specialized neural circuit that positively alters the reward value of lesser-quality foods. Conceivably, a better understanding of the action of this signaling system may provide fresh insights into neural mechanisms for controlling eating and drug-seeking behaviors (Wu, 2006).

Given its prominent role in behavioral adaptation to hunger, the insulin/NPY-like neural network is likely of primary importance to animal evolution. In addition, insulin and NPY family molecules have been found in a wide range of animals from humans to worms. Therefore, the insulin/NPY-like network may be a useful model for studying comparatively how diverse animals have evolved distinct ways of adapting an ancestral neural system to suit their respective lifestyles (Wu, 2006).

Disruption of insulin pathways alters trehalose level and abolishes sexual dimorphism in locomotor activity in Drosophila

Insulin signaling pathways are implicated in several physiological processes in invertebrates, including the control of growth and life span; the latter of these has also been correlated with juvenile hormone (JH) deficiency. In turn, JH levels have been correlated with sex-specific differences in locomotor activity. This study examined the involvement of the insulin signaling pathway in sex-specific differences in locomotor activity in Drosophila. Ablation of insulin-producing neurons in the adult pars-intercerebralis was found to increase trehalosemia and to abolish sexual dimorphism relevant to locomotion. Conversely, hyper-insulinemia induced by insulin injection or by over-expression of an insulin-like peptide decreases trehalosemia but does not affect locomotive behavior. Moreover, this study also showed that in the head of adult flies, the insulin receptor (InR) is expressed only in the fat body surrounding the brain. While both male and female InR mutants are hyper-trehalosemic, they exhibit similar patterns of locomotor activity. These results indicate that first, insulin controls trehalosemia in adults, and second, like JH, it controls sex-specific differences in the locomotor activity of adult Drosophila in a manner independent of its effect on trehalose metabolism (Belgacem, 2006).

In Drosophila, sexual dimorphism has been reported for the number of activity/inactivity phases (start/stops) during locomotion. Feminizing cells (FCs) of the mid-anterior part of the pars intercerebralis (PI), and JH have been implicated in the control of this dimorphism. This study showed that insulin-producing cells (IPCs) are also located in the mid-anterior part of the PI in adult flies, in the same cluster as, but distinct from, the PI neurons termed feminizing cells (FCs). Second, this study showed by conditional genetic ablation that these cells are involved in the control of sex-specific differences in locomotion: flies without IPCs present the same number of start/stops, such that males have a feminine activity profile. Third, perturbation of insulin pathways by mutations affecting the insulin receptor (InR) also has a similar effect on sex-specific locomotion. This result corroborates the idea that control of the number of start/stops by IPCs is mediated by insulin-like peptides and not by another product from these cells. Indeed, the ablation of IPCs removes the cells and everything in them, like putative unrelated but co-expressed peptides or transmitters. However, the finding that the InR mutation leads to a similar phenotype than the IPCs ablation is in accordance with the statement that the number of start/stop might be mediated by the insulin-like peptides. In contrast, hyper-insulinemia induced by insulin injection or by over-expression of the dilp2 gene (hsdilp2) does not affect the number of start/stops in males and thus is not implicated in this sexual dimorphism (Belgacem, 2006).

Another physiological parameter that was investigated is the carbohydrate level in hemolymph. IPCs control the trehalose level at the organismal level via the secretion of insulin analogues. Indeed, a decrease in the insulin level (caused by ablation of IPCs or disruption of insulin signaling with InR mutations) increases the trehalose level, while augmentation of the insulin level (caused by insulin injection or over-expression of dilp2) reduces it. These results clearly show that insulin has an endocrine function in adult Drosophila, as in mammals. In some other insects, insulin and/or insulin analogues have been shown to influence carbohydrate levels, particularly as hypoglycemic hormones (Belgacem, 2006).

This study also reports that the insulin receptor is expressed at the brain periphery in the fat body (FB), as well as in the corpus allatum, the well established site for the JH synthesis. Surprisingly, no InR was found on neuronal cells of the brain. Perhaps InR is not expressed at all in the central nervous system of adult Drosophila, or it was not detected because a heterologous antibody (anti-human) was used to detect it (Belgacem, 2006).

The antibody used was directed against the α-subunit of human insulin receptor, and precisely from the sequence of the third exon, which encodes the insulin-binding domain. A sequence homology performed between this human InR third exon and Drosophila, reveals 36% of homology over 102 amino-acid residues, suggesting that this domain is well conserved. Moreover, the strong detection of the over-expressed InR in the muscle is in accordance with the specificity of the antibody used. Alternatively and in an independent way, physiological approaches have also shown that injection of heterologous insulin (from bovine) is able to activate its invertebrate homologue, both in blowflies as well as in Drosophila, again suggesting a well-conserved domain between different species. Finally, a third argument in accordance with the specificity of the InR is supported by the similar results obtained from the two independent approaches: immunohistological staining analysis and injection of labeled insulin leading to a similar localization in the brain fat body (Belgacem, 2006).

Although InR is expressed both in the head fat body and in the corpus allatum, physiological and behavioral results suggest that the observed effects on sex-specific differences in locomotor activity probably result from disruption of the insulin pathway in the corpus allatum, rather than in the fat body. Indeed, the disruption of the cc-ca gland, by the ablation of the cc, which leads, in males, to a female-like activity profile supports this assumption. Conversely, it is suspected that altered trehalose metabolism phenotype might be due to the distortion of the insulin pathway in the fat body. However, sex-specific differences in locomotor activity under control of signal arising from the fat body could not yet be totally excluded, since two recent studies have suggested that genetic disruption and/or manipulation of the FB affects behavior. The findings that the InR is specifically expressed in FB cells and that the locomotor activity of males lacking InR function is feminized could also correlate with a role for the FB in a sexually dimorphic behavior. Obviously, further experiments, as for instance, tissue-specific targeting of InR disruption, which will require specific GAL4 drivers either in the corpus allatum or fat body, will be necessary for such fine differential dissection (Belgacem, 2006).

The molecular linkage of the insulin pathway to locomotor activity patterns, which also depend on JH levels, remains to be elucidated. Mammalian hydroxymethylglutaryl-CoA reductase (HMGCR), a key enzyme in cholesterol and sterol synthesis, is transcriptionally regulated by the insulin pathway. Drosophila HMGCR is also a central enzyme in the JH biosynthetic pathway and likely plays an important role in JH regulation. Thus, transcriptional regulation of HMGCR by insulin-dependent regulatory elements may link the JH and insulin pathways (Belgacem, 2006).

In conclusion, this study has shown that the insulin signaling pathway is implicated in both males and females in the regulation of trehalose levels, since hypo- and hyper-insulinemia affects both sexes. Moreover, this pathway is also implicated in sex-specific differences in locomotor activity, since perturbations resulting in hypo-insulinemia feminize the locomotor behavior of males, whereas hyper-insulinemia has no effect. Therefore, this new insulin-dependent effect seems to be distinct from the hormonal role of insulin in trehalose level regulation and is likely mediated by either a different intracellular signaling pathway or under control of different tissues. Finally, the identification of the JH target, and more specifically its receptor, is the next crucial step in understanding how brain structures and neurons differentially control sex-specific aspects of locomotor activity (Belgacem, 2006).

IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback

It is generally accepted that the growth rate of an organism is modulated by the availability of nutrients. One common mechanism to control cellular growth is through the global down-regulation of cap-dependent translation by eIF4E-binding proteins (4E-BPs). Evidence is reported for a novel mechanism that allows eukaryotes to coordinate and selectively couple transcription and translation of target genes in response to a nutrient and growth signaling cascade. The Drosophila insulin-like receptor (dINR) pathway incorporates 4E-BP resistant cellular internal ribosome entry site (IRES) containing mRNAs, to functionally couple transcriptional activation with differential translational control in a cell that is otherwise translationally repressed by 4E-BP. Although examples of cellular IRESs have been previously reported, their critical role mediating a key physiological response has not been well documented. These studies reveal an integrated transcriptional and translational response mechanism specifically dependent on a cellular IRES that coordinates an essential physiological signal responsible for monitoring nutrient and cell growth conditions (Marr, 2007).

Coupled transcription and protein synthesis is a hallmark of prokaryotic gene expression. The advantages of such a linked system are well recognized as it provides smooth coordination to ensure that cells respond appropriately to signals such as nutrient availability. A rapid response to such environmental signals also allows for multiple points of regulation and a fine-tuning mechanism for controlling gene expression. In eukaryotic organisms, the compartmentalization of the cell nucleus makes the direct coupling of transcription and translation problematic. Nevertheless, like prokaryotes, the metazoan cell must respond to many external as well as internal signals, and a coupled response would be highly advantageous. However, there is currently little evidence for such a direct linkage, either physical or functional, in metazoans. In attempts to dissect the transcriptional regulatory circuitry of the insulin-like signaling cascade in Drosophila, a potentially new mechanism that functionally links transcription and translation has been identified (Marr, 2007).

Metazoan organisms must strictly control both body and organ size during development. Thus, cell size and cell number are tightly controlled to determine the final size of an animal. One of the cues used in determining growth regulation is nutrient availability. The insulin receptor (INR) and insulin-like growth factor (IGF) receptor pathways have evolved as key sensors of nutrient availability and play an important role in both cell-autonomous and nonautonomous decisions controlling cellular proliferation, cell size determination, and the response to nutrient availability. In Drosophila, this pathway is critical for determining body and organ size as well as metabolic homeostasis and life span. Perhaps most notably, misregulation of this pathway in humans can lead to type 2 diabetes and all of its associated pathologies, which is becoming a rapidly escalating worldwide epidemic (Marr, 2007).

The INR/IGF pathway is highly conserved, with homologs of the key molecular players present in metazoan organisms from flies to humans. The downstream targets of this signaling cascade are thought to separately modulate both transcription and translation to potentiate signals for either growth or stasis. In the presence of insulin or insulin-like peptides, the signaling cascade activates the oncogenic protein kinase Akt. To control RNA synthesis, Akt phosphorylates the Forkhead-box-binding protein (dFOXO) family of transcription factors, sequestering them in the cytoplasm and thus effectively inactivating them. This in turn prevents activated transcription of the dFOXO target genes. In addition, Akt stimulates the modification of the target of rapamycin (TOR) protein, which in turn phosphorylates and inactivates the translation initiation inhibitor eIF4E-binding protein (d4E-BP). In its unphosphorylated and active state, d4E-BP binds to the 7-methyl-guanosine (m7G) cap-binding protein eIF4E. This prevents formation of the translation initiation complex eIF4F, thereby inhibiting cap-dependent translation. This combination of inactivated dFOXO and inactive d4E-BP efficiently drives the cell toward growth and proliferation. Conversely, active dFOXO and d4E-BP conspire to arrest cell growth until the cell receives favorable nutrient and physiological signals to continue proliferation (Marr, 2007).

Drosophila melanogaster has proven to be a valuable model organism for working out the molecular details of this conserved pathway. In the absence of insulin or insulin-like peptides, dFOXO activates the transcription of both the insulin-like receptor (dINR) gene and the gene for Drosophila 4E-BP, establishing a transcriptional signaling loop that sensitizes the cell to receive further nutrient-dependent signals while preventing the cell from proliferating. In order to investigate this intriguing transcriptional feedback control, the start site of transcription for the dINR gene was precisely mapped using a modification of the cap-trapping cDNA synthesis method. This method, which depends on an intact m7G cap for capture of the mRNA, when combined with rapid amplification of five prime (5') cDNA ends (5' RACE) maximizes the yield of full-length 5' untranslated regions (UTRs). The use of this methodology allowed detection of critical UTRs associated with the mRNA that had previously gone undocumented. The dINR gene is actually controlled by a complex set of three distinct promoters (P1, P2, and P3) spread over 38 kb of the Drosophila genome. These combined promoters and associated introns and exons encompass the entire region between the Drosophila E2F gene and the currently annotated dINR gene. This complex control region fills a gap in the genome annotation that contains no other annotated genes or gene predictions (Marr, 2007).

Each of the dINR promoters produces a transcript with a unique and unusually long 5'UTR spliced to a short common exon that is in turn spliced to the first coding exon. The UTR originating from P1 is 1118 bases, the UTR originating from P2 is 419 bases, and the UTR originating from P3 is 485 bases. In contrast, the average 5'UTR in Drosophila is only 256 bases. All three UTRs contain multiple AUG initiator codons upstream of the legitimate INR initiator codon. In the case of the transcript that originates from P1, there are 12 AUGs before the legitimate translational start signal (Marr, 2007).

The DNA sequences immediately upstream of the mapped transcript start sites contain easily recognizable sequences similar to the computationally and biochemically determined common core promoter elements. P1 contains a TATA box, an Initiator element, and a downstream promoter element (DPE). P2 contains a TATA-like box and a DPE but no recognizable Initiator. P3 contains a recognizable Initiator but no recognizable TATA box or DPE. Importantly, a constitutively active form of dFOXO (dFOXO-A3) activates all three promoters in Drosophila Schneider line 2 (S2) cells, and this increased RNA synthesis can produce dINR protein even in the presence of insulin. The transcript originating at P1 is by far the most abundant transcript under both unactivated and activated conditions. P2 is present at an intermediate level, and P3 is a low-abundance transcript. Interestingly, the level of transcription correlates with the number of recognized core promoter elements, illustrating the important role these different elements play in determining the total level of transcription from a gene in both activated and unactivated states (Marr, 2007).

In the animal, all three transcripts are detectable in multiple developmental stages. They are present in whole animal extracts in the same relative order of abundance that is detected in S2 cells (P1 >> P2 > P3). When compared with the Rp49 transcript, a common control transcript that changes little over the stages tested, all three transcripts fluctuate in abundance. Notably, all three transcripts diminish significantly in the L3 larva, a time when the animal is voraciously eating. In contrast, these dINR transcripts peak in the pupae, a time when the animal is fasting and expending much of the energy gained during the larval stage. This observation is consistent with a previous finding that dINR expression is linked to nutrient availability (Marr, 2007).

Strikingly, dINR is not only transcriptionally up-regulated but also robustly translated. Growing S2 cells in the absence of serum and insulin causes a marked decrease in the rate of incorporation of radiolabeled cysteine and methionine consistent with a global decrease in the rate of translation. Despite this slowing of overall translation, dINR protein accumulates in S2 cells. This is detectable by immunoblot of whole cell extracts with antisera raised against the dINR protein. The increase in dINR protein levels is at least partially due to the absence of insulin itself and not another component of serum because the accumulation of dINR protein is inhibited by addition of insulin to media containing insulin-depleted serum. In addition, the increased dINR protein level is most likely due to increased synthesis since serum starved cells contain more radiolabeled receptor that binds to insulin-agarose. This raises the intriguing question of how translation of dINR can proceed in the presence of a quantitatively dephosphorylated, potently active, and up-regulated inhibitor of protein synthesis, d4E-BP. This paradoxical finding that the dINR pathway transcriptionally up-regulates both dINR and d4E-BP combined with the newly discovered unusually long 5'UTRs of these transcripts suggest that perhaps the INR gene engages the translation machinery in an unconventional manner that bypasses the need for eIF4E. A potential d4E-BP resistant internal ribosome entry site (IRES) exists in these Drosophila genes that contain long UTRs, as has been seen in other instances. For example, both the Antennapedia and Ultrabithorax long 5'UTRs contain IRESs, although their physiological role has remained undetermined (Marr, 2007).

As a first test of whether the dINR 5'UTRs also contain an IRES activity, a bicistronic construct, commonly used to assess IRES activity, was generated. The various 5'UTRs of dINR were inserted in both the forward and reverse orientations between the Renilla and firefly luciferase genes. The reverse orientation was used as a spacer length control equivalent. The ratio of Renilla luciferase expression to firefly luciferase expression should provide an indication of the cap-independent translational potential of the various 5'UTRs. Since resistance to d4E-BP is most relevant to this pathway, these experiments were carried out in the presence and absence of a constitutively active form of d4E-BP. Because the Renilla luciferase ORF is the first in the mRNA, it should be uniquely sensitive to inhibition of cap-dependent translation, while the firefly gene expression, if any, should be dependent on internal ribosome entry. The data are expressed as a ratio of the activity in the presence of d4E-BP to the activity in the absence of d4E-BP. Therefore, a number close to 1 indicates that there is no resistance to d4E-BP. In these cell-based assays, the 5'UTR from both P1 and P2 showed significant resistance to d4E-BP (about fourfold better than the reverse orientation in both cases), but only when inserted in the forward direction. Curiously, the 5'UTR from P3 showed unusual resistance to d4E-BP in either orientation. Indeed, the P3 UTR showed a perplexing increase in expression of the firefly ORF in the presence of d4E-BP compared with no UTR in both orientations. This finding reveals a potential limitation of using the bicistronic assays since interfering effects from cryptic promoters, cryptic splicing, or secondary effects of expression of d4E-BP cannot be ruled out with this assay (Marr, 2007).

To circumvent some of the inherent idiosyncrasies of the bicistronic constructs, monocistronic constructs were used that more closely mimic the situation of the endogenous dINR gene. Potential IRES activity esd measured in two complementary ways. First, in a DNA-based transient transfection, either the constitutively active form of d4E-BP or a control protein, green fluorescent protein (GFP), was expressed and resistance to d4E-BP was measure as the ratio of luciferase activity (provided by a second plasmid) in the presence of d4E-BP to the activity in the presence GFP. In this set of experiments, the minimal Antennapedia IRES, a Drosophila 5'UTR known to support cap-independent initiation of translation, was included as a positive control. Under these cell-based assay conditions, the P1 and P2 UTRs again displayed robust resistance to d4E-BP, while P3 and the common exons showed little resistance. Notably, the P2 5'UTR is as efficient as the minimal Antennapedia IRES, and the P1 5'UTR is actually significantly more efficient than the control IRES. Taken together, these two cell-based assays suggest that the 5'UTRs of at least the P1 and P2 transcripts can direct substantial IRES activity, while the P3 UTR appears to have much less if any such activity in S2 cells. Second, to complement these plasmid-based assays and directly investigate the contribution of the UTRs to translation, an RNA-based transfection assay was used. The RNAs contained either a m7G cap or an ApppG cap mimic. Only the 7mG cap allows cap-dependent translation. The ApppG cap stabilizes the transcript but does not allow cap-dependent translation, so it is a direct measure of the contribution of IRES activity. In this assay, the UTRs again showed significant IRES activity. The P1 UTR confers the same activity with or without a m7G cap, indicating a strong IRES activity. The P2 and P3 UTRs also confer cap-independent translation activity, although the level of activity is not equal to UTR plus cap. In contrast, the common exon or nonspecific UTR retains only 20% of their translation potential without the m7G cap. Taken together, these cell-based assays provide encouraging evidence for IRES activity of the dINR 5'UTRs (Marr, 2007).

However, given the well-recognized limitations inherent with using cell-based assays to establish IRES activity, a Drosophila embryo-derived cap-dependant in vitro translation system was used to test more directly the putative IRES activity and more specifically the potential d4E-BP resistance of the INR UTRs. The translation extracts were treated with micrococcal nuclease to destroy the bulk of competing endogenous transcripts so that translation would be largely dependent on exogenously added RNA. As expected, addition of normal capped transcripts results in robust translation from all of the UTR-containing RNAs as well as the common UTR and a short nonspecific UTR control RNA. To test the dependence of translation on eIF4E, exogenous m7G cap analog was added as a competitor. This excess free cap efficiently binds and sequesters the available eIF4E, preventing this essential initiation factor from binding capped RNA, thus effectively blocking the nucleation of the eIF4F complex and cap-dependent initiation. Remarkably, only the transcripts containing the P1, P2, and P3 UTRs are resistant to exogenously added competitor cap analog, whereas the common UTR fragment and the short nonspecific leader are effectively inhibited. This finding strongly suggests that the various dINR-specific UTRs, indeed, provide a cap-independent mechanism of translation initiation. To directly test the resistance of these transcripts to d4E-BP-mediated translation inhibition, recombinant d4E-BP was added to the reactions. Whereas the common exon and control RNAs are efficiently inhibited by this blocker of eIF4E-mediated translation initiation, the P1, P2, and P3 UTR-containing transcripts are highly resistant to d4E-BP. These findings taken together with cell-based assays suggest that, indeed, dINR protein synthesis can proceed via an IRES-mediated eIF4E-independent mechanism of initiation both in vitro and in vivo (Marr, 2007).

What purpose might a cap-independent translation activity serve beyond simple resistance to the active d4E-BP in the absence of insulin? Perhaps by functionally coupling transcription and translation, such a mechanism could serve to amplify the signal received from the insulin receptor pathway. To test this idea, in vitro translation experiments were used. In the absence of miccrococal nuclease treatment, the endogenous transcripts present in the translation extract should effectively compete with the experimental dINR transcripts for limiting amounts of the translation machinery. Advantage was taken of this inevitable competition for translation machinery to test the response of the various UTRs in a situation that may more closely reflect the cellular environment, where multiple variable abundant transcripts must compete for a limited supply of the translational apparatus. Under these competitive conditions, addition of either m7G or d4E-BP actually results in an even more robust increase in translation of the dINR UTR-containing RNAs relative to the unchallenged state. This finding suggests that these RNAs that contain dINR UTRs, and presumably IRES activity, are highly effective at out-competing other transcripts for access to the translational machinery when m7G cap-dependent initiation is inhibited. While the molecular mechanism of 4E-BP resistance of the dINR transcripts have not been unequivocally defined, it is clear that the UTRs allow significant translation in conditions when cap-dependent translation is inhibited (Marr, 2007).

These data allowed formulation of a new model to explain the effects of nutrients and insulin levels on dINR feedback regulation. In times of high nutrients and therefore high insulin-like peptides, both dFOXO and d4E-BP are phosphorylated and inactive. Under these 'rich' conditions, dFOXO is sequestered in the cytoplasm and phosphorylated d4E-BP is unable to interact with eIF4E. This situation allows efficient translation of most cellular transcripts regardless of the mechanism of initiation (cap-dependent vs. cap-independent). In contrast, in low nutrient conditions or in the absence of insulin or insulin-like peptides, both dFOXO and d4E-BP become dephosphorylated and active. Activated dFOXO directs a robust increase in the transcription of both dINR and d4E-BP (among other genes). Additionally, the active and up-regulated d4E-BP effectively inhibits cap-dependent translation, freeing up the protein synthesis machinery to selectively translate IRES-containing transcripts like dINR. These two coordinated mechanisms consequently orchestrate the integration of a specific transcriptional response and simultaneously a translational response that greatly amplifies the signal and sensitizes the cell for detection of small changes in nutrient availability as well as, possibly, developmental and environmental cues (Marr, 2007).

Interestingly, the dFOXO-responsive dINR promoters produce three distinct transcripts. Why such a complex regulatory network? A hint may be that the P3 UTR does not seem to have detectable IRES activity in the S2 cells but shows substantial activity in vitro with extracts derived from whole Drosophila embryos. It is likely that the three transcripts are produced in a tissue- or temporal-specific manner during development, and it is speculated that each may depend on cell-specific IRES trans-acting factors (ITAFs) that are required for activity. This would direct tissues to respond differentially to dINR signaling. In tissues lacking specific ITAFs, the IRES activity would be diminished and the tissue may produce only a moderate level of dINR protein (Marr, 2007).

An interesting parallel was found between mechanisms for reprogramming the gene expression machinery in a cell to respond to physiological cues and the more commonly observed viral takeover of the cellular macromolecular synthesis machinery. When some viruses, such as polio, infect a cell, they target the translation initiation machinery (either eIF4G or 4E-BP) so that there is a switch from cap-dependent synthesis to IRES-dependent synthesis. This leads to a robust and specific stimulation of viral protein synthesis at the expense of most cellular protein synthesis. By the evolution of cellular mechanisms that activate 4E-BP and simultaneously produce transcripts containing cellular IRESs, a critical physiological signaling cascade can evidently adopt a similar mechanism to effectively usurp the macromolecular synthesis machinery to drive cellular physiology in a very specific direction. Indeed, viruses may have merely co-opted the mechanism from cells in the eternal battle between host and virus (Marr, 2007).

Although the initial characterization of the INR transcriptional feedback loop was carried out in Drosophila, a similar regulatory circuit has been found in vertebrates. It is interesting to note that the transcripts for human insulin receptor and IGF-2 receptor remain associated with polysomes when cap-dependant translation is inhibited by poliovirus infection. Although the level of INR mRNA up-regulation by FOXO in mouse muscle cells is only twofold, the levels of INR protein increase much more dramatically (six- to eight-fold), consistent with a coupled transcription/translation mechanism of the signal in vertebrates. It seems likely, given the findings report in this study, that the same type of coupling between the transcriptional program of FOXO proteins and translational control by IRES activity is also occurring in vertebrate systems. Understanding this novel mechanism that couples transcription and translation may provide new insight into disease states such as insulin-resistant type 2 diabetes (Marr, 2007).

MAPK/ERK signaling regulates insulin sensitivity to control glucose metabolism in Drosophila

The insulin/IGF-activated AKT signaling pathway plays a crucial role in regulating tissue growth and metabolism in multicellular animals. Although core components of the pathway are well defined, less is known about mechanisms that adjust the sensitivity of the pathway to extracellular stimuli. In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, which is a hallmark of diabetes. This study presents the results of a genetic screen in Drosophila designed to identify regulators of insulin sensitivity in vivo. Components of the MAPK/ERK pathway were identified as modifiers of cellular insulin responsiveness. Insulin resistance was due to downregulation of insulin-like receptor gene expression following persistent MAPK/ERK inhibition. The MAPK/ERK pathway acts via the ETS-1 transcription factor Pointed. This mechanism permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels (Zhang, 2011).

The insulin signal transduction pathway is regulated by cross-talk from several other signaling pathways. This includes input from the amino-acid sensing TOR pathway into regulation of insulin pathway activity by way of S6 kinase regulating IRS. Signaling downstream of growth factor receptors has also been linked to regulation of insulin signaling. The active form of the small GTPase Ras can bind to the catalytic subunit of PI3K and promote its activity. Expression of a form of PI3K that cannot bind Ras allows insulin signaling, but at reduced levels. The work reported in this study provides evidence for a second mechanism through which growth factor receptor signaling through the MAPK/ERK pathway modulates insulin pathway activity. Transcriptional control of inr gene expression by EGFR signaling may provide a means to link developmental signaling to regulation of metabolism. In this context, a statistically significant correlation wass noted between EGFR target gene sprouty and inr gene expression at different stages during Drosophila development (Zhang, 2011).

Several steps of the insulin pathway can be regulated by phosphorylation. Given that the MAPK/ERK pathway is a kinase cascade, a priori, the possibility of phosphorylation-based interaction between these pathways would seem likely. However, this appears not to be the case. Acute pharmacological inhibition of the MAPK/ERK pathway proved to have no impact on insulin pathway activity. Thus short-term changes in MAPK/ERK pathway activity do not seem to be used for transient modulation of insulin pathway activity. Instead, the MAPK/ERK pathway acts through the ETS-1 type transcription factor Pointed to control expression of the inr gene. Transcriptional control of inr suggests a slower, less labile influence of the MAPK pathway. Taken together with the earlier studies, these findings suggest that growth factor signaling can regulate insulin sensitivity by both transient and long-lasting mechanisms (Zhang, 2011).

Why use both short-term and long-term mechanisms to modulate insulin responsiveness to growth factor signaling? The use of direct and indirect mechanisms that elicit a similar outcome is reminiscent of feed-forward network motifs. Although these motifs are often thought of in the context of transcriptional networks, the properties that they confer are also relevant in the context of more complex systems involving signal transduction pathways. In multicellular organisms, feed-forward motifs are often used to make cell fate decisions robust to environmental noise. The findings suggest a scenario in which a feed-forward motif is used in the context of metabolic control, linking growth factor signaling to insulin responsiveness. In this scenario, growth factor signaling acts directly via RAS to control PI3K activity and indirectly via transcription of the inr gene to elicit a common outcome: sensitization of the cell to insulin. This arrangement allows for a rapid onset of enhanced insulin sensitization, followed by a more stable long-lasting change in responsiveness. Thus a transient signal can both allow for an immediate as well as a sustained response. The transcriptional response also makes the system stable to transient decreases in steady-state growth factor activity. It is speculated that this combination of sensitivity and stability allows responsiveness while mitigating the effects of noise resulting from the intrinsically labile nature of RTK signaling. As illustrated by the data, failure of this regulation in the fat body leads to elevated circulating glucose levels, likely reflecting impaired clearance of dietary glucose from the circulation by the fat body. Maintaining circulating free glucose levels low is likely to be important due to the toxic effects of glucose. In contrast, circulating trehalose, glycogen or triglyceride levels showed no significant change in animals with reduced InR expression, suggesting that these aspects of energy metabolism can be maintained through compensatory mechanisms in conditions of moderately impaired insulin signaling (Zhang, 2011).

Earlier studies have shown that the transcription of the inr gene is under dynamic control. Activation of FOXO in the context of low insulin signaling leads to upregulation of inr transcription, thus constituting a feedback regulatory loop. Thus, InR expression appears to be under control of two receptor-activated cues, which have opposing activities: inr expression is positively regulated by the EGFR-MAPK/ERK module, but negatively regulated by its own activity on FOXO. In the setting of this study, the cross-regulatory input from the MAPK/ERK pathway was found to dominate over the autoregulatory FOXO-dependent mechanism. If conditions exist in which the FOXO-dependent mechanism was dominant, a limited potential for crossregulation by the MAPK/ERK pathway would be expected. Whether Pointed and FOXO display regulatory cooperativity at the inr promoter is an intriguing question for future study (Zhang, 2011).

The Drosophila PGC-1 homologue Spargel coordinates mitochondrial activity to insulin signalling

Mitochondrial mass and activity must be adapted to tissue function, cellular growth and nutrient availability. In mammals, the related transcriptional coactivators PGC-1α, PGC-1β, (Peroxisome proliferator-activated receptor gamma coactivator 1-α and -β) and PRC (PGC-1-related coactivator) regulate multiple metabolic functions, including mitochondrial biogenesis. However, relatively little is known about their respective roles in vivo. This study shows that the Drosophila PGC-1 family homologue, Spargel, is required for the expression of multiple genes encoding mitochondrial proteins. Accordingly, spargel mutants showed mitochondrial respiration defects when complex II of the electron transport chain was stimulated. Spargel, however, was not limiting for mitochondrial mass, but functioned in this respect redundantly with Delg, the fly NRF-2α/GABPα homologue. More importantly, in the larval fat body, Spargel mediates mitochondrial activity, cell growth and transcription of target genes in response to insulin signalling. In this process, Spargel functions in parallel to the insulin-responsive transcription factor, dFoxo, and provides a negative feedback loop to fine-tune insulin signalling. Taken together, these data place Spargel at a nodal point for the integration of mitochondrial activity to tissue and organismal metabolism and growth (Tiefenböck, 2010).

As animals grow, nutrients are taken up, leading to an increase in cellular mass. In this process, mitochondria have critical anabolic and catabolic functions in metabolizing nutrients and in adapting cellular physiology. Therefore, one would expect coordination of mitochondrial mass and activity with growth-promoting pathways and nutrient availability, yet this remains poorly understood. To study mitochondrial biogenesis, most studies focused on individual tissues that show an enormous increase in mitochondrial mass in response to external stimuli, for example, during the formation of the brown adipose tissue (BAT) or perinatal heart maturation. Such studies led to identification and characterization of the PGC-1 family of transcriptional coactivators, which are potent inducers of mitochondrial biogenesis: the founding member PGC-1α (PPARγ coactivator 1; Puigserver, 1998), as well as its homologues PGC-1β and PRC (PGC-1-related coactivator). Mice lacking PGC-1α or PGC-1β showed reduced expression of multiple genes encoding mitochondrial proteins, yet mitochondrial mass and respiration activity were either not or only modestly reduced, depending on the tissue. These mild phenotypes could be because of redundancy. Indeed, RNAi-mediated downregulation of PGC-1β in a PGC-1α-/- background led to strong additive respiration defects in adipocytes. Similarly, mice lacking both PGC-1α and PGC-1β showed defective mitochondrial biogenesis in the heart and the BAT. Although these studies clearly demonstrated critical functions for these proteins in mitochondria-rich tissues, a triple knockout of PGC-1α, PGC-1β and PRC has not been published; therefore it remains unclear whether PGC-1s are generally required for basal mitochondrial mass (Tiefenböck, 2010).

PGC-1 family members drive mitochondrial biogenesis through coactivation of nuclear transcription factors, including nuclear respiratory factor-1 and -2 (NRF-1 and NRF-2), estrogen-related receptor-α (ERRα) and YY1, to enhance the expression of genes encoding mitochondrial proteins. Accordingly, NRF-1 and ERRα are known to be functionally important for PGC-1s to stimulate mitochondrial mass. Similarly, NRF-2 promoter-binding sites were required for coactivation by PGC-1α and PRC in certain genes, and PRC can coactivate NRF-2β. However, it is not known whether NRF-2 is required for PGC-1's effect on mitochondrial function, or whether NRF-2 is controlled through other factors (Tiefenböck, 2010).

In flies and mammals, insulin signalling and TOR (target of rapamycin) function are known to link cellular growth and metabolism to nutrition. Recent data showed that mammalian TOR (mTOR) and mitochondrial oxidative capacity are tightly linked, and mTOR inhibition reduced the association of PGC-1α with the transcription factor YY1, leading to lower expression of several genes encoding mitochondrial proteins. These data demonstrate that mTOR is a crucial regulator of mitochondrial function in mammals. In flies, nutrient starvation and subsequent inhibition of insulin signalling led to reduced expression of multiple genes encoding mitochondrial proteins. However, a direct role of TOR has not been addressed in these studies. Moreover, the majority of genes encoding mitochondrial proteins were not responsive to TOR inhibition in Drosophila cells. Therefore, mechanisms in addition to TOR must exist to adapt mitochondrial mass and function to nutrient availability (Tiefenböck, 2010).

This study investigated the role of the Drosophila PGC-1 homologue Spargel/CG9809 in the control of mitochondrial biogenesis and activity. The Drosophila genome encodes a single PGC-1 homologue, thus providing a system in which PGC-1 function can be analysed without interfering redundancy. Focused was placed on the larval fat body, the functional equivalent of the mammalian adipose tissue and liver. In this tissue, many genes encoding mitochondrial proteins were expressed in a nutrient-sensitive manner. The larval fat body is, therefore, ideal to study how mitochondrial mass and activity are coordinated with cellular metabolism and nutrient supply. This study shows that Spargel is required for proper expression of most genes encoding mitochondrial proteins. When complex II of the electron transport chain is stimulated, spargel mutants show respiration defects. Remarkably, Spargel is not required for basal mitochondrial mass, but becomes limiting in the absence of Delg, the fly NRF-2α homologue. Moreover, from these and previous experiments, Spargel and Delg were shown to function in two different pathways, each regulating mitochondrial in response to nutrients. Finally, the question how insulin signalling affected mitochondria was addressed; Spargel was found to be required for the stimulation of mitochondrial respiration, and to a large extent for the transcriptional control mediated by insulin signalling, including that of genes encoding mitochondrial proteins. Moreover, insulin signalling induces Spargel gene expression and protein levels, and Spargel mediates a negative feedback loop on insulin signalling. These data demonstrate a critical role for Spargel in the coordination of mitochondria with nutrients, and thus in cellular metabolism (Tiefenböck, 2010).

The D. melanogaster genome encodes a single PGC-1 homologue, CG9809 (Gershman, 2007). Sequence alignments have shown that the N-terminal acidic domain that mediates transcriptional coactivation for mammalian PGC-1s, and the C-terminal arginine-serine-rich and RNA recognition domains are highly conserved in the fly protein, yet its cellular function has not been addressed. To test whether CG9809 is a functional PGC-1 homologue in flies, mutants that have a P-element insertion (KG08646) in the 5' UTR were examined. In comparison with controls (precise excision of the P-element), homozygous mutant larvae had a strong reduction in CG9809 mRNA levels, adults were viable, and females were sterile. Both males and females had a 25% reduction in wet weight, which correlated with reduced protein, lipid, glycogen and trehalose levels per animal. When normalized to body weight, reduced lipid and glycogen levels were observed in adult males, demonstrating metabolic defects. When external structures that are derived from imaginal discs were analysed, such as wings or legs, no large size defects were detected in CG9809 mutants. These animals have, therefore, a lean phenotype, prompting use of the name 'Spargel' German for 'asparagus', and the KG08646 allele as srl1. To test the specificity of this allele, a second P-element insertion (d04518, termed srl2), was tested; this showed the same phenotypes as srl1 and transgenic flies were created carrying a genomic rescue construct, which suppressed all mutant phenotypes. As another gene, CG31525, is located within the first intron of Spargel, a fly line was created expressing a Spargel cDNA under the control of the UAS promoter. When driven using heat-shock Gal4, UAS-Srl also suppressed the mutant phenotypes, demonstrating that loss of Spargel function was responsible for the observed phenotypes. Finally, a trans-heterozygous combination of srl1 with Df(3R)ED5046, a deficiency that deletes the Spargel locus, led to a further reduction in Spargel transcript levels, yet it did not lead to a further decrease in adult weight compared with srl1 homozygous mutant animals. Taken together, srl1 is a strong hypomorphic allele, showing a ~75% reduction in mRNA levels (Tiefenböck, 2010).

Mitochondrial mass and activity must adapt to cellular growth rates and nutrient availability, yet factors involved were poorly described. This study showed that Drosophila Spargel is critical for proper expression of genes encoding mitochondrial proteins, and that it mediates a link to the nutrient-sensitive insulin signalling pathway. In the larval fat body, Spargel was required for proper mitochondrial respiration when complex II was stimulated, both under normal and insulin signalling stimulated conditions. These data support the interpretation that the control of mitochondria represents an ancestral function of the PGC-1 proteins family. Importantly, it was shown that Spargel is not a master regulator of mitochondrial biogenesis, but becomes limiting in this respect in the absence of Delg. Furthermore, ectopic expression of Spargel was not sufficient to drive mitochondrial abundance, which contrasts mammalian data, where PGC-1's are potent stimulators of mitochondrial mass. Mammalian PGC-1 proteins are induced by external stimuli, including cold exposure in BAT or exercise in muscle tissues, thus adapting the cellular physiology in response to such stimuli (Puigserver, 2003). To test a similar function for Drosophila Spargel, larvae were exposed to cold shocks, and respiration rates from dissected fat bodies were measured by complex I stimulation. Although cold exposure led to reduced respiration, this response was not altered in the spargel mutant. Thus this function is not conserved in flies, which correlates with previous findings that the Drosophila fat body resembles the mammalian white adipose tissue, which is not involved in thermoregulation. Furthermore, genes involved in gluconeogenesis, β-oxidation and lipogenesis, all functions linked to mammalian PGC-1 proteins, were not reduced in the spargel mutant, at least not in the larval fat body. No altered lipid levels were detected in spargel mutant larvae, thus these functions might be vertebrate-specific. Potentially, these differences can be explained by the finding that Spargel lacks the canonical LXXLL motifs, which mediate binding to multiple transcription factors for mammalian PGC-1s . Spargel however contains a conserved C-terminal FXXLL motif, which could mediate transcription factor binding, yet such interactors remain elusive (Tiefenböck, 2010).

In mammalian cells, overexpression of PGC-1α led to increased expression of NRF-2α/GABPα, and NRF-2 binding sites in the promoters of mitochondrial transcription factors TFB1M and TFB2M were required for coactivation by PGC-1α and PRC. Similarly, PRC was shown to coactivate NRF-2β-dependent transcription, thus it was assumed that PGC-1 proteins and NRF-2 would function in the same pathway. Very importantly, these studies focused on individual genes, but not on mitochondrial function and mass, thus it is still unclear whether NRF-2 is functionally required for PGC-1 proteins in respect to mitochondrial mass. Although Drosophila Spargel and Delg may share many putative target genes, these factors function in parallel pathways in respect to mitochondrial mass, morphology and OXPHOS activity (Tiefenböck, 2010).

The respiration defects in spargel single mutants can be explained by reduced expression of genes involved in oxidative phosphorylation. Spargel mutants however did not show a reduction in mitochondrial abundance, which appears surprising. In wild-type larvae, the fat body does not attract tracheoles for gas exchange, and shows physiological low oxygen levels, suggesting low rates of mitochondrial respiration. Indeed, compared to other larval tissues, a low inner-mitochondrial membrane potential and reduced oxygen consumption was detected. This suggests that oxidative phosphorylation might not be a critical function of the fat body mitochondria. Rather, this tissue releases lipids and amino acids, in particular proline and glutamine, providing energy sources for other tissues. Since proline and glutamine are synthesized from the mitochondrial TCA cycle intermediate 2-oxoglutarate, it is proposed that such a function is rate limiting for mitochondrial mass in the fat body. Delg but not Spargel was required for proper expression of genes involved in proline and glutamine metabolism, thus explaining the mitochondrial abundance defect in the delg mutant, and the absence of such defects in the spargel mutant (Tiefenböck, 2010).

Spargel and Delg are distinct in respect to upstream signalling: Spargel is functionally required for insulin signalling, which was not seen for Delg. In contrast, Delg, but not Spargel, is required for Cyclin D/Cdk4 to stimulate mitochondrial abundance. Thus the data provided genetic evidence that Spargel and Delg represent two different pathways, each regulating mitochondria in response to nutrients. Although several recent reports have demonstrated a functional link between insulin signalling and PGC-1 proteins in mammalian cells, the interaction appears to be complex, and is most likely tissue and/or context dependent: Some studies showed that PGC-1 proteins were required for insulin signalling, whereas other studies found an inhibitory function for PGC-1α. In flies, the insulin signalling pathway is well conserved, and since Spargel is the only PGC-1 protein, Drosophila is an ideal organism to study a functional interaction between insulin signalling and PGC-1 proteins in vivo (Tiefenböck, 2010).

Spargel is required for insulin signalling stimulated growth, functioning downstream of Dp110, but presumably independently of Akt, and balances signalling activity through a negative-feedback loop. Very remarkably, Spargel mediates ~40% of the transcriptional control in response to insulin signalling, emphasizing a major function in the control of cellular growth and metabolism. Moreover, these data suggest that Spargel functions independently of dFoxo, therefore representing a novel transcriptional output of insulin signalling in the larval fat body. This appears to contradict recent microarray data, which showed repressed Spargel mRNA levels upon expression of an activated form of dFoxo in culture Drosophila cells. The discrepancy could be due to tissue-inherent differences. In this study, INR expression led to an increase in Spargel mRNA and protein abundance, further suggesting that Spargel has a direct role within the insulin signalling pathway. Future experiments are required to establish a molecular mechanism how Spargel function and subcellular localization is mediated by insulin signalling components. Analogous to the current results, reduced PGC-1α protein levels were observed upon knockdown of the insulin receptor InsRβ in mammalian cells, suggesting a conserved mechanism between flies and mammals. Thus the data are further genetic evidence for a functional interaction between PGC-1 proteins and insulin signalling. Although flies and mammals are several hundred millions apart in evolution, Spargel and its mammalian counterparts PGC-1α, PGC1-1β and PRC have conserved functions in respect to mitochondria and insulin signalling. In the future, this will allow use of Drosophila as a powerful system to understand regulatory circuitries that control homeostasis of cellular metabolism (Tiefenböck, 2010).

Identification of a novel gene, anorexia, regulating feeding activity via insulin signaling in Drosophila melanogaster

Feeding activities of animals, including insects, are influenced by various signals from the external environment, internal energy status, and physiological conditions. Full understanding of how such signals are integrated to regulate feeding activities has, however, been hampered by a lack of knowledge about the genes involved. This study identified an anorexic Drosophila melanogaster mutant (GS1189) in which the expression of a newly identified gene, Anorexia (Anox; CG33713), is mutated. In Drosophila larvae, Anox encodes an acyl-CoA binding protein with an ankyrin repeat domain that is expressed in the cephalic chemosensory organs and various neurons in the central nervous system (CNS). Loss of its expression or disturbance of neural transmission in Anox-expressing cells decreased feeding activity. Conversely, overexpression of Anox in the CNS increased food intake. It was further found that Anox regulates expression of the insulin receptor gene (dInR); overexpression and knockdown of Anox in the CNS, respectively, elevated and repressed dInR expression, which altered larval feeding activity in parallel with Anox expression levels. Anox mutant adults also showed significant repression of sugar-induced nerve responses and feeding potencies. Although Anox expression levels did not depend on the fasting and feeding states cycle, stressors such as high temperature and desiccation significantly repressed its expression levels. These results strongly suggest that Anox is essential for gustatory sensation and food intake of Drosophila through regulation of the insulin signaling activity that is directly regulated by internal nutrition status. Therefore, the mutant strain lacking Anox expression cannot enhance feeding potencies even under starvation (Ryuda, 2011).

Insulin production and signaling in renal tubules of Drosophila is under control of tachykinin-related peptide and regulates stress resistance

The insulin-signaling pathway is evolutionarily conserved in animals and regulates growth, reproduction, metabolic homeostasis, stress resistance and life span. In Drosophila seven insulin-like peptides (DILP1-7) are known, some of which are produced in the brain, others in fat body or intestine. This study shows that DILP5 is expressed in principal cells of the renal tubules of Drosophila (see Three Overviews of the Drosophila Malpighian Tubule for information on Malpighian tubule structure) and affects survival at stress. Renal (Malpighian) tubules regulate water and ion homeostasis, but also play roles in immune responses and oxidative stress. This study investigated the control of DILP5 signaling in the renal tubules by Drosophila tachykinin peptide (DTK) and its receptor Tachykinin-like receptor at 99D during desiccative, nutritional and oxidative stress. The DILP5 levels in principal cells of the tubules are affected by stress and manipulations of DTKR expression in the same cells. Targeted knockdown of DTKR, DILP5 and the insulin receptor dInR in principal cells or mutation of Dilp5 resulted in increased survival at either stress, whereas over-expression of these components produced the opposite phenotype. Thus, stress seems to induce hormonal release of DTK that acts on the renal tubules to regulate DILP5 signaling. Manipulations of S6 kinase and superoxide dismutase (SOD2) in principal cells also affect survival at stress, suggesting that DILP5 acts locally on tubules, possibly in oxidative stress regulation. These findings are the first to demonstrate DILP signaling originating in the renal tubules and that this signaling is under control of stress-induced release of peptide hormone (Söderberg, 2011).

The insulin-signaling pathway is evolutionarily conserved in multicellular animals and insulin-like peptides (ILPs) regulate growth, reproduction and metabolism and play important roles in stress resistance and regulation of life span. In Drosophila genetic ablation of cells in the brain producing ILPs, or mutations in the ILP receptor (dInR) and other insulin signaling components, lead to an increase in stress tolerance and extension of life span at the expense of fertility and body size. Also carbohydrate and lipid homeostasis is affected by these manipulations. Seven Drosophila ILPs (DILP1-7) have been identified and some of these are expressed in the brain, others in fat body or intestine. Although much has been learned about insulin signaling downstream of the insulin receptor, it is not clear how the production and release of DILPs is regulated in adult Drosophila in response to nutritional or stress signals. Nutritional sensing appears to take place in adipose tissue, the fat body, and recently it was shown that there is a humoral link between the fat body and insulin-producing cells (IPCs) in the brain. Thus, availability of nutrients sensed by the fat body is an important factor in regulation of DILP release. In addition recent evidence suggest that the IPCs can sense glucose levels autonomously (Söderberg, 2011).

It is likely that hormonal or neural signals also regulate production and release of DILPs by IPCs of the adult insect, as has been shown to be the case in pancreatic beta-cells in mammals. However, such hormones have not yet been identified in the fly, although recently neurons expressing, short neuropeptide F, GABA or serotonin were suggested as regulators of DILP production in IPCs of the brain. The role of DILPs in stress responses is intriguing and this study sought to investigate hormonal signaling pathways that mediate regulation of release of DILPs during stress in Drosophila (Söderberg, 2011).

For nutritional and osmotic stress one possible hormonal route is signaling from endocrine cells of the intestine. The intestine could provide further sensors to monitor metabolic status and it has been shown that midgut endocrine cells in insects release peptide hormone at starvation. A few candidate peptide hormones have been identified in endocrine cells of the Drosophila intestine. This study focused on peptides encoded by the gene Tachykinin (Tk, Dtk or CG14734), the five Drosophila tachykinin-related peptides DTKs, and the role of their receptors in regulation of DILPs in the fly. The reason for this focus is that a novel set of cells was detected that produce DILP5 and also express one of the two known receptors for DTKs (Söderberg, 2011).

This study shows that the main epithelial cells of the renal tubules (Malpighian tubules), the principal cells, express both DILP5 and the DTK receptor DTKR, suggesting that these insulin-producing cells are targets of circulating DTKs. Indeed, it was found that DTK signaling regulates levels of DILP5 in principal cells under nutritional stress. Since the renal tubules are not innervated, DTK can only reach them as a circulating hormone, likely to be released from the intestine (Söderberg, 2011).

In Drosophila the renal tubules display high metabolic activity and play roles, not only in water and ion transport, but also in oxidative stress, detoxification and immune responses. Encouraged by this and by the likely importance of insulin signaling in the physiology of the kidneys of mammals, the roles were investigated of DILP5 signaling locally in the renal tubules. Interference with the expression levels of DTKR, DILP5, dInR and some further components of the insulin-signaling pathway in principal cells during metabolic and oxidative stress all lead to altered lifespan. Furthermore, knockdown of superoxide dismutase (SOD2) in principal cells leads to decreased lifespan at desiccation and oxidative stress, suggesting a possible link between insulin signaling and oxidative stress responses. It is proposed that insulin signaling in the tubules may be part of an autocrine regulation of renal function that in turn is controlled by hormonal DTK signaling from the intestine at metabolic and oxidative stress (Söderberg, 2011).

This study has identified the renal tubules as a novel site of insulin production and signaling in Drosophila. The principal cells of these tubules produce DILP5 and express the ubiquitous DILP receptor, dInR. From these findings it is suggested that DILP5 may signal locally within the epithelium of the renal tubules. This local DILP signaling appears to be under hormonal regulation during desiccative, nutritional and oxidative stress by means of the peptide DTK acting on the receptor, DTKR, localized on the principal cells. These findings, that diminished DTKR, DILP5 and dInR extend life span, suggest an involvement of this signaling pathway in tubules in desiccation, nutritional and oxidative stress responses in adult Drosophila. Finally, manipulations of dS6K, 4E-BP and SOD (SOD2) in principal cells altered life span of flies at stress supporting that insulin signaling acts within the tubules, probably in regulation of oxidative stress responses. Interestingly, the signaling within the renal tubules affects the survival of the whole organism as shown also for mitochondrial function in tubules at oxidative stress (Söderberg, 2011).

The roles of DILPs in stress resistance and regulation of life span are well established in Drosophila, but hormonal mechanisms for regulation of production and release of DILPs in IPCs of adult flies have not been reported. Thus this demonstration of DTKs acting on IPCs in the renal tubules is a first identification of a hormonal factor regulating DILP release in adult insects. Interestingly, there is evidence for actions of tachykinins on IPCs also in mammals: the tachykinin substance P has been shown to increase insulin secretion from the pancreas of rat and pig and this effect is reversed in the diabetic rat (Söderberg, 2011 and references therein).

Since the renal tubules are not innervated, peptide receptors in this tissue can only be activated by hormonal messengers. One source of hormonal DTKs in Drosophila is a population of endocrine cells in the intestine (midgut) located close to the attachment of the renal tubules. In locust and cockroach similar cells have been identified and it was shown that at starvation tachykinin-related peptide was released into the circulation (Söderberg, 2011).

Renal tubules in insects have been primarily investigated with respect to their function in water and ion transport and several peptide hormones have been implicated in the control of diuresis. The current findings suggest that peptide hormones that target the renal tubules may play roles other than in direct regulation of diuresis. The Drosophila renal tubules express an impressive array of genes and combined with experimental analysis it is suggestive that this tissue partakes in detoxification processes, oxidative stress, dietary osmotic stress and immune responses (Söderberg, 2011 and references therein).

How does DTK signaling to the renal tubules produce a response that affects sensitivity to desiccation and starvation? The DTK signal may be a general metabolic stress signal that reaches the renal tubules. In these experiments this stress signaling is amplified with the over-expression of DTKR in principal cells and diminished by its knockdown leading to changes in lifespan. The role of DTK may be to regulate factors in principal cells involved in local metabolism, oxidative stress resistance or immune responses at the cost of decreased life span when in over-drive. One such a factor may be DILP5. Both in Drosophila and C. elegans immune response genes are expressed in the intestine (including renal tubules in the fly) and recent work has shown that these genes are under control of insulin signaling. In Drosophila the DILP signaling pathway is involved in infection-induced wasting (loss of energy stores) where reduced signaling leads to reduction in pathology (Söderberg, 2011).

Also oxidative stress resistance is linked to insulin signaling in Drosophila. Superoxide dismutases (SOD) are key enzymes protecting proteins from reactive oxygen species and are thought to be regulated by insulin signaling: SOD activity is elevated in chico (dInR substrate) mutants of Drosophila and Daf-2 mutants of C. elegans. Also in yeast insulin-signaling mutations affect lifespan via SOD. The knockdown of Sod2 (encoding MnSOD), but not Sod1, in renal tubules decreased lifespan at desiccation and oxidative stress in Drosophila. Thus, it is possible that DILP signaling in tubules target mitochondrial SOD2 and affects resistance to oxidative stress. Interestingly, diminishing oxidative stress resistance via Sod2 locally in the principal cells of Drosophila renal tubules is sufficient to shorten the lifespan of the fly during stress. This is similar to findings in a study of genetical impairment of a mitochondrial inner membrane ATP/ADP exchanger in the same cells (Söderberg, 2011),

In conclusion, this study presents evidence for DTK controlled insulin signaling in the renal tubules of Drosophila being important for survival at metabolic and oxidative stress. The findings of this study may suggest an autocrine regulatory loop within the tubules with a role in renal function. Local signaling within Drosophila renal tubules has previously been demonstrated with endogenously produced tyramine and nitric oxide, that regulate chloride permeability and innate immune responses, respectively. It is possible that the insulin signaling in the renal tubules is part of the epithelial immune system or oxidative stress defense via SOD, but it cannot be excluded that the dInRs on principal cells regulate DILP5 production or release and that additional DILP5 targets are located outside the renal tubules (Söderberg, 2011).

Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues

Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on losing access to these signals. This study demonstrates that differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot properly differentiate. An insulin peptide produced by somatic cells immediately outside of the stem cell niche was found to act locally to promote somatic differentiation through Insulin receptor (InR) activation. These results indicate that there is a local 'differentiation' niche which upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, it was demonstrated that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, this study shows that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that local production of positive and negative InR signals regulate the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation is actively induced by signaling (Amoyel, 2016).

This study shows that PI3K/Tor activity is required for the differentiation of somatic stem cells in the Drosophila testis. Additionally, a 'differentiation' niche was identified immediately adjacent to the stem cell niche that, through the local production of Dilps, leads to the upregulation of PI3K/Tor activity in early CySC daughters and to their commitment to differentiation. The secretion of ImpL2 by CySCs antagonizes the initiation of differentiation in CySCs by blocking available Dilps in the stem cell niche. As a result, CySCs receive little free Dilp ligands. However, as their daughters move away from the hub, they encounter increasing levels of Dilps and decreasing levels of ImpL2, which leads to the upregulation of PI3K/Tor signaling and proper somatic cell differentiation. The fact that ImpL2 is upregulated by the main self-renewal signal (i.e., JAK/STAT) in CySCs leads to a model accounting for the spatial separation of the stem cell niche and the differentiation niche (Amoyel, 2016).

The results are consistent with a model in which autocrine or paracrine production of Dilp6by early cyst cells serves as a differentiation niche in the testis, defining where in the tissue upregulation PI3K/Tor signaling - a prerequisite for differentiation - occurs. This differentiation niche is critical for somatic development because stem cell markers like Zfh1 are maintained in the absence of signals like PI3K/Tor. Notably, JAK/STAT activity is not expanded outside of the niche upon somatic loss of PI3K/Tor signaling, suggesting that differentiation signals play a critical role in downregulating stem cell factors. Intriguingly, recent studies in the Drosophila ovary have identified a differentiation niche in this tissue: autocrine Wnt ligands produced by somatic support escort cells regulate escort cell function, proliferation and viability. Taken together, these studies reveal that at least in Drosophila gonads, there is a defined region immediate adjacent to the stem cell niche where autocrine production of secreted factors induces the differentiation of somatic cells, which in turn promote development of the germ line (Amoyel, 2016).

Several studies have examined the role of insulin signaling in gonadal stem cells. In both testes and ovaries, systemic Dilps have been shown to affect stem cell behavior. In both tissues, nutrition through regulation of systemic insulin controls the proliferation rate of GSCs. The current data showing that Akt1, Dp110 or Tor mutant CySC clones proliferate poorly are consistent with these findings and indicate that basal levels of insulin signaling are required for the proliferation and/or survival of both stem cell pools in the testis. This work also demonstrates that production of a secreted Insulin binding protein ImpL2 by CySCs reduces available Dilps in the stem cell niche, and ImpL2 in the niche milieu should reduce insulin signaling in GSCs and CySCs. While these data seemingly contradict the results that insulin is required for GSC maintenance, a model is suggested in which low constitutive levels of insulin signaling are required for stem cell proliferation and that higher levels are required to induce stem cell differentiation. (Amoyel, 2016).

Prior reports have found that both male and female flies with reduced Insulin or Tor activity are sterile, and the results presented in this study suggest that this is due at least in part to a lack of somatic cell differentiation. The results indicate that Dilp6, the IGF homolog, plays a local role in CySC differentiation, but acts redundantly with other presumably systemic factors, suggesting that both constitutive and nutrient-responsive inputs control CySC differentiation. Indeed, this study shows that in addition to controlling the proliferation of stem cells, systemic insulin is required for their differentiation, as the poorly proliferative Akt1, Dp110 or Tor mutant CySC clones do not differentiate and eventually die by apoptosis. This combination of reduced proliferation and increased apoptosis may explain why other studies suggest that Tor is required for self- renewal in GSCs; indeed prior reports indicate that while Tor mutant GSCs are lost, hyper-activation of Tor leads to faster loss of GSCs through differentiation and recent work indicates that lineage-wide Tor loss blocks the differentiation of GSCs. The use of hypomorphic alleles enabled a genetic separation of the proliferative effects and differentiation requirements of PI3K and Tor in CySCs. Finally, there is evidence that PI3K/Tor activity promotes differentiation of stem cells in gonads in mammals, suggesting that these findings may reflect a conserved role of Tor activity in promoting germ cell differentiation, both through autonomous and non- autonomous mechanisms involving somatic support cells. Moreover, it seems likely that Tor activity may be a more general requirement for the differentiation of many stem cell types, as increased PI3K or Tor has been shown to induce differentiation in many instances. In particular, mouse long term hematopoietic stem cells are lost to differentiation when the PI3K inhibitor Pten is mutated, while Drosophila intestinal stem cells differentiate when Tor is hyperactive due to Tsc1/2 complex inactivation. Moreover, inhibition of Tor activity by Rapamycin promotes cellular reprogramming to pluripotency, while cells with increased Tor activity cannot be reprogrammed, suggesting a conserved role for Tor signaling in promoting differentiated states (Amoyel, 2016).

Insulin signaling in the peripheral and central nervous system regulates female sexual receptivity during starvation in Drosophila

Many animals adjust their reproductive behavior according to nutritional state and food availability. Drosophila females for instance decrease their sexual receptivity following starvation. Insulin signaling, which regulates many aspects of insect physiology and behavior, also affects reproduction in females. This study shows that insulin signaling is involved in the starvation-induced reduction in female receptivity. More specifically, females mutant for the insulin-like peptide (dilp5) were less affected by starvation compared to the other dilp mutants and wild-type flies. Knocking-down the insulin receptor, either in all fruitless-positive neurons or a subset of these neurons dedicated to the perception of a male aphrodisiac pheromone, decreased the effect of starvation on female receptivity. Disrupting insulin signaling in some parts of the brain, including the mushroom bodies even abolished the effect of starvation. In addition, Fruitless-positive neurons in the dorso-lateral protocerebrum and in the mushroom bodies co-expressing the insulin receptor were identified. Together, these results suggest that the interaction of insulin peptides determines the tuning of female sexual behavior, either by acting on pheromone perception or directly in the central nervous system (Lebreton, 2017).

Drosophila females need nutrients to produce eggs and a nutrient rich substrate to lay their eggs. When food is scarce it would therefore be beneficial for flies to decrease their sexual behavior and to focus on food searching instead. On the other hand, female flies can store sperm and use it several days later when conditions are suitable. It could therefore be optimal for females to remain receptive for short periods of food deprivation. Several insulin peptides produced in specific spatiotemporal patterns acting through one single receptor enables a fine-scale regulation of behaviors in response to changes in physiology. The expression of the different dilps is differentially affected by food quality or food deprivation. For instance, both starvation and dietary restriction reduce the expression of dilp5 but increase the expression of dilp6, while the expression of dilp2 is not affected by either condition. The results suggest that DILP5 might be involved in the decrease of receptivity during non-feeding stages. Indeed, dilp5 mutant females were less affected by starvation than other dilp mutants. The effect of the lack of DILP5 was no longer observed in the simultaneous absence of DILP2 and DILP3. Although, background mutation effects cannot be completely ruled out, this suggests that DILP5 might interact with other DILPs to finely tune female sexual receptivity (Lebreton, 2017).

Insulin is known to act on the olfactory system to modulate odor sensitivity after feeding. Moreover, normal InR expression in Or67d-expressing (Fruitless-positive) OSNs is necessary for fed females to be attracted to a blend of food odors and cVA, a pheromone promoting sexual receptivity. The results suggest that insulin signaling in Fruitless-positive neurons, and more specifically in Or67d OSNs may decrease sexual receptivity during starvation (Lebreton, 2017).

Fruitless-positive cells other than pheromone-sensing neurons can also be involved. Different Fruitless-positive cells in the protocerebrum were found that strongly express InR. First of all, a large number of Kenyon cells in the calyx of the mushroom bodies express both Fruitless and the insulin receptor. Additionally, one pair of neurons was found with somata located in the anterior dorso-lateral protocerebrum. It was not possible to trace any processes from these somata, and thus it is not known what neuropils they innervate. However, the fact that InR immunostaining was observed in Fruitless neurons, most of which were Kenyon cells, corroborate the behavioral results. Indeed, the sexual receptivity of females in which insulin signaling was knocked down in the mushroom bodies was not affected by starvation. Interestingly, the mushroom bodies are not required for virgin females to be receptive, suggesting that these structures may regulate the activity of neuronal networks inducing sexual receptivity. However, this result must be take with caution, given the fact that the Gal4 line that were used to target the mushroom bodies also drive expression to some extent in other brain tissues. Further experiments will be necessary to confirm that the mushroom bodies are indeed responsible for this effect (Lebreton, 2017).

Insulin signaling not only modulates neuronal activity in adults but also shapes neuronal networks during development. The effects observed in this study may therefore be the consequence of a developmental defect of specific neuronal circuitry rather than a direct effect of insulin on these neurons during starvation. However, Fruitless-positive neurons being required for females to be receptive, fed females would be expected to be unreceptive if the disruption of insulin signaling had altered the connectivity of these neurons during development, which was not the case. This suggests that insulin acts on these neurons during adult stage to modulate sexual receptivity. This is different for the mushroom bodies, which are not necessary for females to be receptive. Knocking down InR specifically during development or specifically in adults will be necessary to disentangle these two possible modes of action of insulin (Lebreton, 2017).

In contrast with Fruitless neurons and the mushroom bodies, no effect was observed of the corpora allata in the insulin-dependent control of sexual receptivity, whereas these structures have been linked to the development of receptivity in virgin females. This result should however be taken with caution, considering the behavioral variability displayed by the different transgenic lines, which would have prevented observing of subtle changes. Nonetheless, the results suggest that the structures that generate behaviors (such as the corpora allata) and those modulating these behaviors (for example the mushroom bodies) can be different and the underlying mechanisms uncoupled (Lebreton, 2017).

Taken together, Drosophila flies adjust their sexual behavior to match their nutritional state. Together with other hormonal pathways, insulin regulates some aspects of sexual activity, both after food intake and after a period of starvation. The results suggest that specific insulin peptides regulate female receptivity, possibly by acting on pheromone perception at the periphery or directly in the central nervous system. Indeed, the mushroom bodies probably play a major role in the insulin-dependent effect of starvation on female sexual receptivity. The next step will be to untangle the specific neuronal circuitry involved (Lebreton, 2017).

Protein Interactions

The glycosylation of the Drosophila insulin receptor (InR) has been compared to that of the rat insulin receptor by means of an examination of the binding of receptors to the lectins wheat germ agglutinin, Concanavalin-A, and lentil lectin. Although rat insulin receptors bind and are specifically eluted from all three lectins, only a small fraction of the InR (< 5%) is retained on wheat germ agglutinin. In contrast, the InR binds strongly to Concanavalin-A and lentil lectin and is recovered from lentil lectin columns after elution with alpha-methyl-mannoside. The pattern of lectin binding indicates that glycosylation of the InR and rat insulin receptors differs, with the InR containing primarily high mannose-type oligosaccharides. After lectin chromatography, the InR exhibits an elevated level of basal autophosphorylation and kinase activity, which can be restored to a low level by incubation with 0.5 mM dithiothreitol (DTT). DTT does not, however, affect ligand-stimulated kinase activity. The ability of low concentrations of DTT to deactivate the InR kinase suggests that, like the mammalian receptor, beta-subunit thiols may be involved in regulation of conformational changes between activated and unactivated receptor states. Interestingly, DTT-induced deactivation of the InR is blocked by preincubation with an antipeptide antibody against the carboxy-terminal domain of the InR. This suggests that the InR carboxyl terminus undergoes a conformational change during the activation-inactivation cycle of the kinase, which can be sterically hindered by the antibody. Conformational changes in this region of the mammalian receptor have been observed, and these data suggest that features of the insulin receptor activation mechanism have been substantially conserved during evolution (Marin-Hincapie, 1995).

Chimeric receptors encoding either the whole or a portion of the cytoplasmic domain of the Drosophila insulin receptor (InR) with the extracellular domain of the human insulin receptor (IR) were expressed either transiently in COS cells or stably in Chinese hamster ovary cells and compared with the wild-type human IR. All three receptors bind insulin equally and exhibit an insulin-activated tyrosine kinase activity. The ability of the Drosophila cytoplasmic domain to mediate the tyrosine phosphorylation of insulin receptor substrate 1, stimulate cell proliferation, and activate MAP kinase is indistinguishable from that of the human IR. The chimeric Drosophila receptors do not bind more phosphatidylinositol 3-kinase (see Phosphotidylinositol 3 kinase 92E) than the human IR, despite containing a C-terminal extension with potential tyrosine phosphorylation sites in the motif recognized by the SH2 domain of this enzyme. Thus, the essential signal-transducing abilities of the IR appear to have been conserved from invertebrates to mammals, despite the considerable differences in the sequences of these receptors (Yamaguchi, 1995).

The InR proreceptor [M(r) 280 kDa] is processed proteolytically to generate an insulin-binding alpha subunit [M(r) 120 kDa] and a beta subunit [M(r) 170 kDa] with a protein tyrosine kinase domain. The InR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension that contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage that leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90, and a free 60 kDa C-terminus) from the inr gene. These subunits assemble into mature InR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation for both types of beta subunits; in turn, the phosphorylation allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of InR have different signaling potentials. Loss of function mutations in the InR gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, renders pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of InR appears to be required in the embryonic epidermis and nervous system among organ systems, since development of the cuticle, as well as the peripheral and central nervous systems are affected by InR mutations (Fernandez, 1995).

Stimulation of the activity of protein kinase C by pretreatment of cells with phorbol esters was tested for its ability to inhibit signaling by four members of the insulin receptor family, including the human insulin and insulin-like growth factor-I receptors, the human insulin receptor-related receptor, and the Drosophila insulin receptor. Activation of overexpressed protein kinase Calpha results in a subsequent inhibition of the ligand-stimulated increase in antiphosphotyrosine-precipitable phosphatidylinositol 3-kinase mediated by the kinase domains of all four receptors. This inhibition varies from 97% for the insulin receptor-related receptor to 65% for the Drosophila insulin receptor. In addition, the activation of protein kinase Calpha inhibits the in situ ligand-stimulated increase in tyrosine phosphorylation of the GTPase-activating protein-associated p60 protein as well as Shc mediated by these receptors. The mechanism for this inhibition was further studied in the case of the insulin-like growth factor-I receptor. Although the in situ phosphorylation of insulin-receptor substrate-1 and p60 by this receptor is inhibited by prior stimulation of protein kinase Calpha, the in vitro tyrosine phosphorylation of these two substrates by this receptor is not decreased by prior stimulation of the protein kinase Calpha in the cells that served as a source of the substrates. Finally, the insulin-like growth factor-I-stimulated increase in cell proliferation was found to be inhibited by prior activation of protein kinase Calpha. These results indicate that the ability of activated protein kinase Calpha to antagonize signaling by the human insulin receptor is shared by the other members of the insulin receptor family despite their considerable differences in amino acid sequence. Moreover, the present study shows that this antagonism is exerted at a very early step, the initial tyrosine phosphorylation of three distinct endogenous substrates. Finally, the present study indicates that this inhibition is not caused by an increased Ser/Thr phosphorylation of these two substrates (Danielsen, 1996).

A monoclonal antibody has been produced that immunoprecipitates 58- and 53-kDa proteins that are rapidly tyrosine phosphorylated in insulin-treated cells. These proteins can also be tyrosine phosphorylated in vitro by the isolated human insulin receptor. Increased tyrosine phosphorylation of these proteins is also observed in cells expressing a transforming chicken c-Src (mutant Phe-527) and in cells with the activated tyrosine kinase domains of the Drosophila insulin receptor, human insulin-like growth factor I receptor, and human insulin receptor-related receptor. P58/53 does not appear to associate with either the GTPase activating protein of Ras (called GAP) or the phosphatidylinositol 3-kinase by either co-immunoprecipitation experiments or in Far Westerns with the SH2 domains of these two proteins. Since p58/53 does not appear, by immunoblotting, to be related to any previously described tyrosine kinase substrate such as the SH2 containing proteins SHC and the tyrosine phosphatase Syp, the protein was purified in sufficient amounts to obtain peptide sequence. This sequence was utilized to isolate a cDNA clone that encodes a previously uncharacterized 53-kDa protein that, when expressed in mammalian cells, is tyrosine phosphorylated by the insulin receptor (Yeh, 1996).

Drosophila contain an insulin receptor homolog, encoded by the InR gene located at position 93E4-5 on the third chromosome. The receptor protein is strikingly homologous to the human receptor, exhibiting the same alpha2beta2 subunit structure and containing a ligand-activated tyrosine kinase in its cytoplasmic domain. Chemical mutagenesis was used to induce mutations in the inr gene. Six independent mutations that lead to a loss of expression or function of the receptor protein have been identified. These mutations are recessive, embryonic, or early larval lethals, but some alleles exhibit heteroallelic complementation to yield adults with a severe developmental delay (10 days), growth-deficiency, female-sterile phenotype. Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase. The growth deficiency appears to be due to a reduction in cell number, suggesting a role for InR in regulation of cell proliferation during development. The phenotype is reminiscent of those seen in syndromes of insulin-resistance or IGF-I and IGF-I receptor deficiencies in higher organisms, suggesting a conserved function for this growth factor family in the regulation of growth and body size (Chen, 1996).

The Drosophila insulin receptor (InR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins that binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric InR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulates tyrosine autophosphorylation of both the human insulin receptor and hDIR, and both receptors mediate tyrosine phosphorylation of Shc and activate mitogen-activated protein kinase. IRS-1 is required by the human insulin receptor to activate PI 3-kinase and p70s6k (see Drosophila RPS6-p70-protein kinase), whereas hDIR associates with PI 3-kinase and activates p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the InR possesses additional signaling capabilities when compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells (Yenush, 1996).

Like the mammalian insulin receptor, the Drosophila insulin receptor (INR)1 is a tetramer formed by two alpha subunits and two beta subunits. INR alpha and beta subunits are synthesized together as a proreceptor precursor, proteolytically processed, and linked together by disulfide bonds. The alpha subunits, with a molecular mass of 110-120 kDa, are extracellular and contain the ligand binding domains that are capable of binding mammalian insulin with a Kd of 15 nM. The beta subunits traverse the plasma membrane and have an insulin-stimulated tyrosine kinase in the cytoplasmic portion. DNA sequence analysis and expression of the INR beta subunit in mammalian and Drosophila cells indicate that the INR beta subunit is larger than its mammalian homolog and exhibits an apparent molecular mass of ~180 kDa. The increased mass is due to the presence of a 400-amino acid carboxyl-terminal extension. However, the majority of INR beta subunits are processed to 92/102-kDa forms in Drosophila embyros and some cell lines, the difference being due to proteolytic cleavage of the carboxyl-terminal extension. Both truncated and full-length beta subunits are autophosphorylated on tyrosine residues in response to insulin binding (Marin-Hincapie, 1999 and references therein).

The 400-amino acid carboxyl-terminal extension of the beta INR contains clusters of motifs known to be involved in the interaction with SH2 and PTB domain-containing proteins, suggesting a role for this domain in signaling through interaction with other signaling molecules. Interestingly, four tyrosines are found in 'hybrid' amino acid motifs in which residues amino-terminal to each tyrosine form the motif NP X Y, resembling known PTB domain binding sites, and residues carboxyl-terminal to the same tyrosines form the motifs YXXM, YMXM, or YXLLD -- all known to be involved in binding to SH2 domains. Thus, tyrosines 1993 and 2030 appear in the motif SXNPXYXX M; tyrosine 2009 is part of S X NPXYMXM, and tyrosine 1969 appears in the sequence SDNPXYRLLD. Whether these motifs serve to bind SH2 or PTB domain-containing proteins upon tyrosine phosphorylation and whether one is preferred over the other is not clear. The cytoplasmic domain of the INR expressed in cells lacking IRS-1 has been shown to bind PI3-kinase. However, a similar construct expressed in Chinese hamster ovary cells that contain IRS-1 fails to do so. Since a significant percentage of the INR beta subunit undergoes tissue- or stage-specific proteolytic processing in Drosophila embryos to remove the carboxyl-terminal extension and once it is removed it appears not to be phosphorylated, its role in signal transduction by the INR is not clear. Therefore, the signaling capacity conferred by the beta INR carboxyl-terminal extension has been explored by expressing either full-length or truncated INR beta subunit forms in mammalian cells and determining the effect on protein-protein interactions and cell growth (Marin-Hincapie, 1999 and references therein).

In order to explore the role of the 400 AA extention in INR function, mammalian expression vectors encoding either the complete INR beta subunit (beta-Myc) or the INR beta subunit without the carboxyl-terminal extension (betaDelta) were constructed, and the membrane-bound beta subunits were expressed in 293 and Madin-Darby canine kidney (MDCK) cells in the absence of the ligand-binding alpha subunits. beta-Myc and betaDelta proteins are constitutively active tyrosine kinases of 180 and 102 kDa, respectively. INR beta-Myc co-immunoprecipitates a phosphoprotein of 170 kDa identified as insulin receptor substrate-1 (IRS-1, Flipper or Chico), whereas INR betaDelta does not, suggesting that the site of interaction is within the carboxyl-terminal extension. IRS-1 is phosphorylated on tyrosine to a much greater extent in cells expressing INR beta-Myc than in parental or INR betaDelta cells. Despite this, a variety of PTB or SH2 domain-containing signaling proteins, including IRS-2, mSos-1, Shc, p85 subunit of phosphatidylinositol 3-kinase, SHP-2, Raf-1, and JAK2, are not associated with the INR beta-Myc.IRS-1 complex. Overexpression of INR beta-Myc and betaDelta kinases confers an equivalent increase in cell proliferation in both 293 and Madin-Darby canine kidney cells, indicating that this growth response is independent of the carboxyl-terminal extension. However, INR beta-Myc-expressing cells exhibit enhanced survival, relative to parental and betaDelta cells, suggesting that the carboxyl-terminal extension, through its interaction with IRS-1, plays a role in the regulation of cell death (Marin-Hincapie, 1999).

Thus, overexpression of constitutively active INR beta and betaDelta receptors in 293 and MDCK cells promotes cell proliferation, indicating that the INR can engage the mammalian proliferation pathways. The equivalent proliferative responses induced by INR beta-Myc and betaDelta kinases suggests that the growth-promoting function of the INR in these cells is independent of the carboxyl-terminal extension. In contrast, cells expressing the full-length INR beta subunit exhibit significantly enhanced survival as compared with cells expressing the betaDelta INR. Relative to the parental 293 and MDCK cells, the INR beta-Myc and betaDelta proteins confer somewhat different behavior; beta-Myc clearly promotes survival in 293 cells, whereas betaDelta more dramatically accelerates cell death in MDCK cells. Nonetheless, a clear difference in the behavior of cells expressing the full-length or truncated INR beta subunits is evident in both backgrounds. Despite the presence of a juxtamembrane NPXY motif predicted to interact with IRS-1 in both beta-Myc and betaDelta proteins, IRS-1 is not highly phosphorylated in betaDelta cells. This suggests that the carboxyl-terminal extension of the INR beta subunit is required for sustained association and phosphorylation of IRS-1. This persistent IRS-1 phosphorylation distinguishes beta-Myc from betaDelta cells and may be of primary importance in promoting cell survival. Without this sustained interaction, cell death may actually be accelerated, as observed in MDCK cells transfected with the INR betaDelta kinase (Marin-Hincapie, 1999).

IRS-1 that is bound to the INR beta subunit is phosphorylated on tyrosine; however, no evidence has been found for increased association of PI3-kinase or other candidate signaling molecules with this complex. Therefore, the mechanism whereby this association leads to increased cell survival is unclear at present. Interestingly, a recent report demonstrates that expression of a truncated IRS-1 containing only the pleckstrin homology and phosphotyrosine binding domains, without any tyrosine phosphorylation sites, mediates PI3-kinase and phosphotyrosine-independent signals that contribute to the regulation of cell survival and apoptosis. IRS-1 that is bound to the carboxyl-terminal extension of INR in 293 and MDCK cells may have similarly activated pathways that promote cell survival in the absence of PI3-kinase activation (Marin-Hincapie, 1999 and references therein).

Thus, two isoforms of an activated INR beta subunit have been expressed in mammalian cells, and a functional difference between them has been demonstrated. The data presented here indicate that the stimulation of cell proliferation by INR is mediated by the kinase domain independent of the carboxyl-terminal extension. In contrast, the carboxyl-terminal extension mediates an interaction with IRS-1 and influences cell survival. Since an IRS homolog is present in Drosophila, this may reflect an inherent function of the INR which, in flies, is modulated by tissue- or stage-specific processing of the receptor. These data also suggest that in mammalian cells, persistent localization of IRS-1 to membranes via the interaction of IRS-1 with receptors and/or persistent tyrosine phosphorylation generates signals independent of association with PI3-kinase (Marin-Hincapie, 1999 and references therein).

Insulin receptor substrate (IRS) proteins are phosphorylated by multiple tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream signaling pathways. The Drosophila insulin receptor (InR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that InR might not require an IRS protein to accomplish its signaling functions. However, a cDNA encoding Drosophila IRS (Chico, but referred to in this study as dIRS) has been obtained and one for Chico in a Drosophila cell line has also been demonstrated. Like mammalian IRS proteins, the N-terminal portion of Chico contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with Chico in COS-7 cells, a chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic domain of InR) mediates the insulin-stimulated tyrosine phosphorylation of Chico. Mutating the juxtamembrane NPXY motif markedly reduces the ability of the receptor to phosphorylate Chico. In contrast, the NPXY motifs in the C-terminal extension of InR are required for stable association with Chico. Coimmunoprecipitation experiments demonstrate insulin-dependent binding of Chico to phosphatidylinositol 3-kinase and SHP2. However, interactions with Grb2, SHC, or phospholipase C-gamma were not detected. Taken together with published genetic studies, these biochemical data support the hypothesis that Chico functions directly downstream from the insulin receptor in Drosophila (Poltilove, 2000).

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy

Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, a high conservation in Dg-Dys interaction between human and Drosophila is demonstrated. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR (Shcherbata, 2007).

The Dg-Dys binding interface is highly conserved in humans and Drosophila. Both proteins are required for oocyte cellular polarity and interact in this process. Futhermore, mutants of both Dg and Dys genes show symptoms observed in muscular dystrophy. Reduction of Dg and Dys proteins results in age-dependent mobility defects. Eliminating Dg and Dys specifically in mesoderm derived tissues reveals that these proteins are required for muscle maintenance in adult flies: age-dependent muscle degeneration was observed in mutant tissues. Dg-Dys complex is also required for neuron path-finding and has both cell autonomous and non-cell autonomous functions for this process. Further, in neuronal path-finding process Dg interacts with InR and an SH2/SH3-domain adapter molecule Nck/Dock (Shcherbata, 2007).

Animal models have been used efficiently in muscular dystrophy studies. Some of the models are naturally occurring mutations (mdx-mouse, muscular dystrophy dog, cat and hamster), others have been generated by gene targeting. However, the regulation and the control of Dg-Dys complex are not understood, and no successful therapeutics exist yet for muscular dystrophies. Recently developed models for muscular dystrophy exist in C. elegans and zebrafish. In C. elegans Dys mutant, the transporter snf-6 that normally participates in eliminating acetylcholine from the cholinergic synapses, is not properly localized, resulting in an increased acetylcholine concentration at the neuromuscular junction and muscle wasting (Kim, 2004). The function of Dys in neuromuscular junctions has been addressed in Drosophila. These results bring up the possibility that muscular dystrophies in humans might also at least partly be attributed to the altered kinetics of acetylcholine transmission through neuromuscular junctions (Shcherbata, 2007).

Drosophila acts as a remarkably good model for age-dependent progression of muscular dystrophy. Dg and Dys reduction in Drosophila show age-dependent muscle degeneration and lack of climbing ability. It is tempting to speculate that the common denominator between different defects observed in Dg-Dys mutants in Drosophila and C. elegans is defective cellular polarity. The defects observed in C. elegans could be due to a defect in polarization of a cell, which will generate a neuromuscular junction that leads to miss-targeted snf-6. Similarly, Drosophila Dg-Dys complex is required for cellular polarity in the oocyte. In addition, neural defects observed are plausibly due to polarity defects in the growing axon (Shcherbata, 2007).

Similar to neuronal defects observed in human muscular dystrophy patients, neuronal defects were also found in Drosophila Dg and Dys mutant brains. In vertebrate brains, Dg affects neuronal migration (Montanaro, 2003; Qu, 2004) possibly through interaction of neurons with their glial guides. The neuronal migration and process outgrowth have been shown to require supportive input from glial cells and involve the formation of adhesion junctions along the length of the soma. Also, the outgrowth of the leading process involves rapid extension and contraction over the length of the glial fiber. Disruption of the cytoskeletal organization within the neuron, either of actin filaments, has been shown to inhibit glial-mediated neuronal migration. The glial function in this process is less well studied (Shcherbata, 2007).

Drosophila photoreceptor path-finding provides an excellent system for genetic dissection of neuronal outgrowth and target recognition. During the formation of the nervous system, newly born neurons send out axons to find their targets. Each axon is led by a growth cone that responds to extracellular axon guidance cues and chooses between different extracellular substrates upon which to migrate. Recent work has also identified a variety of intracellular signaling pathways by which these cues induce cytoskeletal rearrangements, but the proteins connecting signals from cell surface receptors to actin cytoskeleton have not been clearly determined. Dg is a good candidate for linking receptor signaling to the remodeling of the actin cytoskeleton and thereby polarizing the growth cone. Perturbation of Dg-Dys complex causes phenotypes that resemble Nck/Dock-Pak-Trio axon path-finding phenotypes, suggesting that Dg may be one of the key players in Nck/Dock signaling pathway for axon guidance and target recognition in Drosophila (Shcherbata, 2007).

Interestingly, Insulin receptor-tyrosine kinase (InR) mutants also show similar phenotypes to those of Nck/Dock signaling in photoreceptor axon path-finding and these two proteins show genetic and biochemical interactions. These data have led to speculations of mammalian InR acting in conjunction with Nck/Dock pathway in learning, memory and eating behavior. The current data now add Dg-Dys complex to this pathway; similar to what is seen in the case of Dg and Dys photoreceptor mutants, InR mutants show no obvious defects in patterning of the photoreceptors. However, the guidance of photoreceptor cell axons from the retina to the brain is aberrant. Furthermore, genetic and biochemical evidence suggests that InR function in axon guidance involves the Dock-Pak pathway rather than the PI3K-Akt/PKB pathway. Independently, biochemical interaction between Nck/Dock and Dg has been reported supporting the hypothesis that InR, Dg and Nck/Dock interaction regulates Dg-Dys complex. Furthermore, Dg interacts genetically with InR and Dock in photoreceptor axon path-finding. Since Dys interacts with Dg but not with InR and Dock, it is tempting to speculate that Dg can selectively interact with either Dys or InR and Dock. One possibility is that the tyrosine kinase activity of InR could regulate the Dg-Dys interaction by tyrosine phosphorylation in the Dg-Dys binding interphase. This tyrosine phosphorylation could prohibit the Dg-Dys interaction and thereby result in rearrangements in the actin cytoskeleton. Alternatively, other components observed in Dg-Dys complex might be involved in this regulation. However, it is also possible that potential polarity defects in the Dg mutant axons result in defective InR membrane localization. Interestingly, in another cell type, the Drosophila oocyte, InR, Dg and Dys also show similar phenotypes. In addition, insulin-like growth factors (IGF) and InR are important in maintaining muscle mass in vertebrates. Further connection of InR to Dg-Dys complex comes from experiments showing that muscle specific expression of IGF counters muscle decline in mdx-mice. The work presented in this study is the first demonstration of genetic interaction between Dg and InR. Future biochemical studies should unravel the molecular mechanism of this interaction (Shcherbata, 2007).

Dg-Dys complex is required both in neural and in targeting glial cells for correct neuronal axon path-finding in Drosophila brain. These data reveal that Dg-Dys complex also has a non-cell autonomous effect on axon path-finding and suggest that Dg-Dys-controlled ECM both from neuron and glial cells regulate neuronal axon path-finding. Further experiments are required to reveal whether long-range Laminin fibers are involved in this process, as has been shown in epithelial planar polarity, or whether glial processes are observed in close proximity to the neural growth cone. Interestingly, similar phenotypes are observed with Integrin mutants, suggesting that, as in planar polarity, Integrin and Dg-Dys complex might act in concert to regulate the process of ECM organization that will regulate the cytoskeleton of the cells involved (Shcherbata, 2007).

Taken together, the phenotypes caused by Drosophila Dg and Dys mutations are remarkably similar to phenotypes observed in human muscular dystrophy patients, and therefore suggest that functional dissection of Dg-Dys complex in Drosophila should provide new insights into the origin and potential treatment of these fatal neuromuscular diseases. As a proof of principle, using Drosophila as a model, InR has now been identified as a signaling pathway that genetically interacts with Dg. Future studies are directed to unravel the molecular mechanism of Dg and InR-Dock interactions in invertebrates as well as vertebrates (Shcherbata, 2007).

Nonautonomous regulation of Drosophila midgut stem cell proliferation by the insulin-signaling pathway

Drosophila adult midgut intestinal stem cells (ISCs) maintain tissue homeostasis by producing progeny that replace dying enterocytes and enteroendocrine cells. ISCs adjust their rates of proliferation in response to enterocyte turnover through a positive feedback loop initiated by secreted enterocyte-derived ligands. However, less is known about whether ISC proliferation is affected by growth of the progeny as they differentiate. This study shows that nutrient deprivation and reduced insulin signaling results in production of growth-delayed enterocytes and prolonged contact between ISCs and newly formed daughters. Premature disruption of cell contact between ISCs and their progeny leads to increased ISC proliferation and rescues proliferation defects in insulin receptor mutants and nutrient-deprived animals. These results suggest that ISCs can indirectly sense changes in nutrient and insulin levels through contact with their daughters and reveal a mechanism that could link physiological changes in tissue growth to stem cell proliferation (Choi, 2011).

Previous studies have focused on responses of ISC proliferation to enterocyte death, delineating a positive feedback mechanism by which ligands secreted from dying enterocytes activate ISC proliferation. The data propose a model of additional regulation where cell contact between ISCs and newly formed enteroblasts acts to inhibit ISC proliferation through a negative feedback loop (see Cell contact regulates ISC proliferation) (Choi, 2011).

Nutrient deprivation leads to decreased ISC proliferation rates and clones containing fewer cells than clones made in animals fed a rich diet. However, it is unclear why these clones fail to eventually reach the same size as wild-type clones. One possibility is that nutrient-deprived midguts contain fewer cells. Therefore, the number of cells that each ISC needs to generate to maintain tissue homeostasis would be smaller. A second possibility is built on the observation that turnover and production of 8n and 16n enterocytes is reduced in animals fed a poor diet, and this could result in the depletion of a source of promitotic ligands, thereby decreasing the need for a stem cell to divide (Choi, 2011).

Protein deprivation and reduced insulin signaling leads to an increase in the number of lower ploidy enterocyte daughters per midgut, suggesting that endoreduplication in the midgut is regulated by nutrition. Because enterocyte turnover is reduced in nutrition-deprived animals, it raises the intriguing possibility that 8n cells act to inhibit the growth and endoreduplication of 4n cells into mature enterocytes through an as-yet-unidentified signal. These similarities between nutrient-deprived clones and dInR mutant clones suggest that the effects of nutrition may be mediated in part through the insulin-signaling pathway. Consistent with a role for nutrition and the insulin-signaling pathway in growth and endoreduplication, constitutive activation of dInR in ISC clones led to enterocytes with significantly higher ploidy than normal. Interestingly, these clones were smaller than wild-type, suggesting that excessive or prolonged contact between enterocytes and ISCs may also play a role in the regulation of ISC proliferation (Choi, 2011).

The findings raise the as-yet-unexplored possibility that germ-line stem cell and neuroblast stem cell daughters might also nonautonomously regulate stem cell proliferation. When both the ISC and the enteroblast were mutant for dInR, a further increase in cell cycle arrest was observed, suggesting an autonomous role for insulin signaling in the regulation of ISC proliferation (Choi, 2011).

Significantly higher levels of DE-cadherin were found between both dInR mutant enteroblast and wild-type ISCs and dInR mutant enteroblasts and dInR mutant ISCs, demonstrating that the insulin-signaling pathway regulates the stability of the adherens junction. The results are striking because, in the ovary and testis, loss of dInR signaling in the germ-line stem cell niche leads to a decrease rather than an increase in DE–cadherin at the adherens junction (Choi, 2011).

The data presented in this study demonstrate that the enteroblast can nonautonomously regulate the rate of ISC proliferation. How might this be achieved? One possibility is that the enteroblast inhibits ISC proliferation by providing a short-range inhibitory signal whose effect is removed as the ISC and enteroblast separate. A second possibility is that separation of ISCs and enteroblasts leads to the release from a cellular compartment of a factor that can drive proliferation. The ideal candidate is β-catenin, which is not only a member of the adherens junction but also a transcriptional activator, which is required for ISC proliferation (Choi, 2011).

Recently, ISCs and enteroblast number were examined under protein-poor conditions in old animals expressing green fluorescent protein driven by the escargot promoter (esg-GFP), which is thought to be specific to ISCs and enteroblasts. A decrease in esg-GFP–positive cells was observed in 16- to 17- and 20- to 21-d-old animals fed a poor diet, leading to the conclusion that ISC maintenance is regulated by a protein-poor diet. In contrast, this study did not observe a decrease in ISC number in females fed a protein-poor diet. Presumably, the modest decrease in GFP-positive cells observed by the previous study was due to loss of the excess enteroblasts seen in aging midguts, which is consistent with recently published work showing that insulin-signaling mutants can suppress this aging phenotype (Choi, 2011).

A putative tyrosine phosphorylation site of the cell surface receptor Golden goal is involved in synaptic layer selection in the visual system.

Golden Goal (Gogo) is a cell surface protein that is crucial for proper synaptic layer targeting of photoreceptors (R cells) in the Drosophila visual system. In collaboration with the seven-transmembrane cadherin Flamingo (Fmi), Gogo mediates both temporary and final layer targeting of R-cell axons through its cytoplasmic activity. However, it is not known how Gogo activity is regulated. This study shows that a conserved Tyr-Tyr-Asp (YYD) tripeptide motif in the Gogo cytoplasmic domain is required for photoreceptor axon targeting. Deleting the YYD motif is sufficient to abolish Gogo function. The YYD motif is shown to be a phosphorylation site, and mutations in the YYD tripeptide impair synaptic layer targeting. Gogo phosphorylation results in axon stopping at the temporary targeting layer, and dephosphorylation is crucial for final layer targeting in collaboration with Fmi. Therefore, both temporary and final layer targeting strongly depend on the Gogo phosphorylation status. Drosophila Insulin-like receptor (DInR) has been reported to regulate the wiring of photoreceptors. This study shows that insulin signaling is a positive regulator, directly or indirectly, of YYD motif phosphorylation. These findings indicate a novel mechanism for the regulation of Gogo activity by insulin signaling-mediated phosphorylation. It is proposed that a constant phosphorylation signal is antagonized by a presumably temporal dephosphorylation signal, which creates a permissive signal that controls developmental timing in axon targeting (Mann, 2012).

The YYD tripeptide (Tyr1019-Tyr1020-Asp1021) is conserved among invertebrate and vertebrate species and has a crucial role: deleting it is sufficient to completely abolish Gogo function. YYD is a phosphorylation site and the phosphorylation status of Gogo is critical for both temporary and final layer targeting. A model is proposed in which Gogo is phosphorylated during the first targeting step. A prolonged phosphorylation during the mid-pupal stage prevents R8 axons from extending to their final layer (Mann, 2012).

The current experiments show that it is the dephosphorylated form of Gogo that is the most active. The non-phospho-Gogo is functional during R8 targeting as it could be used to rescue the gogo minus mutant. By contrast, phosphomimetic Gogo does not rescue the mutant phenotype. This mechanism resembles the molecular regulation of Robo activity in that dephosphorylated Robo shows the most activity in mediating repulsive signals during embryonic CNS axon guidance. Additionally, protein inactivation upon phosphorylation, although relatively rare, has been reported in biochemical pathways; for instance, the inactivation of the transcriptional co-activator Yorkie by Warts and others (Mann, 2012).

Gogo function was abolished by removing the YYD site (GogoδYYD) or by mimicking phosphorylation (GogoDDD). Both forms result in a very strong adhesiveness and in the stopping of growth cones at the M1 layer, suggesting the involvement of phosphorylation in the first targeting step. It was postulated that two independent pathways could be activated depending on the phosphorylation status, resulting in either normal M3 layer targeting or M1 stopping, and their activation could be mutually exclusive (Mann, 2012).

Although dephosphorylated Gogo enables the axons to leave the M1 layer, it is not clear whether in a physiological situation phosphorylation contributes to adhesiveness to the M1 layer. Phosphorylation might not entirely be necessary, as axons expressing only the dephosphorylated GogoFFD can still recognize the M1 layer. However, there might be more fine-tuning defects, such as in the location of the synapses along the R8 axons between the M1 and M3 layers (Mann, 2012).

Defects in dephosphorylation can also result in mistargeting of the M3 layer. This is supported by the fact that phospho-Gogo does not show the proper cooperation with Fmi during final layer targeting. Normally, for a proper Gogo-Fmi collaboration, colocalization is required. However, Fmi can colocalize with both phospho- and non-phospho-Gogo, suggesting that the phosphorylation status is important for signal transmission and not for the interaction with Fmi (Mann, 2012).

Another molecule known to interact with Gogo, Hts (the Drosophila homolog of adducin 1) was shown to bind to Gogo and to play a role in guiding photoreceptors. However, this physical interaction occurs independently of the YYD site (Mann, 2012).

Further evidence for the role of Gogo phosphorylation during R8 targeting comes from the experiments in which the Gogo phosphorylation status was genetically modulated, looking for kinases and phosphatases that might modulate the Gogo phosphorylation status. The Drosophila genome contains a relatively small number of tyrosine kinases and phosphatases. This study focused on proteins that are expressed in the brain, that show a transmembrane localization and are implicated in axon guidance. Since Gogo YYD motif phosphorylation is involved in the characteristic overexpression phenotype, it was convenient to screen for the suppression or enhancement of M1 blobs when a kinase or phosphatase was co-overexpressed with gogo. From a number of genes tested (Abl, Src42A, Src64B, drl, Egfr, dinr, Lar, Ptp69D, eya) dinr was identified as a possible regulator of Gogo phosphorylation. All other tested kinases were excluded from a detailed analysis because the overexpressed genes either did not enhance/suppress the gogo gain-of-function phenotype, resulted in extensive cell death, or caused a severe axon guidance phenotype that was difficult to distinguish from a cell death phenotype (Mann, 2012).

It is striking that GogoDDD causes a much stronger adhesiveness to the M1 layer than Gogo phosphorylated by overexpressed DInR. A possible explanation is that, unlike GogoDDD, DInR-dependent phosphorylation in this case is not complete. Alternatively, there are redundant mechanisms that further modulate Gogo phosphorylation. Therefore, only a small proportion of photoreceptors stop at the M1 layer and the majority of axons form blob-like structures (Mann, 2012).

The cues that a growing axon encounters can be divided into instructive or permissive. Instructive cues usually have a restricted expression pattern and guide the axon by providing either attractive or inhibitory information to the growth cone. Permissive signals steer in response to instructive or are needed to detect and respond to extracellular guidance cues (Mann, 2012).

These findings confirm the intriguing possibility that insulin signaling modulates axon guidance. It is difficult to imagine that DInR transduces a typical guidance cue: the only known ligands for DInR, the DILPs, are secreted into the circulatory system and thus cannot provide a directional cue. Rather, insulin signaling could orchestrate the guidance signals coming from instructive directional cues. Insulin signaling could be essential for ensuring the correct wiring of the nervous system by influencing the phosphorylation of a regulator of photoreceptor axon guidance, Gogo. Gogo phosphorylation provides a signal that enhances the adhesive interaction with the M1 layer, whereas dephosphorylation could provide a permissive signal that allows the axon to leave the M1 layer and project to the M3 targeting layer (Mann, 2012).

The state of phosphorylation of a protein at any moment, and thus its activity, depends on the relative activities of the protein kinases and phosphatases that modify it. This suggests that a Gogo dephosphorylation mechanism exists. It would be rewarding to identify the phosphatase that mediates Gogo dephosphorylation and thereby constitutes an essential regulator of Gogo activity. Preliminary genetic studies of several candidates, including Lar and Ptp69D, have not revealed any genetic interaction so far. In summary, a mechanism is proposed whereby the activity of the axon guidance receptor Gogo is regulated by phosphorylation mediated by DInR and dephosphorylation mediated by an as yet unknown phosphatase. This may provide insight into how developmental timing is coordinated in neuronal circuit wiring through a phosphorylation-dephosphorylation mechanism (Mann, 2012).


The Drosophila insulin receptor InR is similar to its mammalian counterpart in deduced amino acid sequence, subunit structure, and ligand-stimulated protein tyrosine kinase activity. The function of this receptor in D. melanogaster is not yet known. However, a role in development is suggested by the observations that levels of insulin-stimulated kinase activity and expression of InR mRNA are maximal during Drosophila midembryogenesis. In this study, a 2.9-kilobase (kb) cDNA clone corresponding to both the InR tyrosine kinase domain and some of the 3' untranslated sequence was used to determine the tissue distribution of InR mRNA during development. Two principal mRNAs of 11 and 8.6 kb hybridize with the InR cDNA in Northern (RNA) blot analysis. The abundance of the 8.6-kb mRNA increases transiently in early embryos, whereas the 11-kb species is most abundant during midembryogenesis. A similar pattern of expression has been determined by Northern analysis, using an InR genomic clone. In situ hybridization revealed InR transcripts in the ovaries of adult flies, in which the transcripts appear to be synthesized by nurse cells for eventual storage as maternal RNA in the mature oocyte. Throughout embryogenesis, InR transcripts are ubiquitously expressed, although after midembryogenesis, higher levels are detected in the developing nervous system. Nervous system expression remains elevated throughout the larval stages and persists in the adult, in which the cortex of the brain and ganglion cells are among the most prominently labeled tissues. In larvae, the imaginal disc cells exhibit comparatively high levels of InR mRNA expression. The broad distribution of InR mRNA in embryos and imaginal discs is compatible with a role for InR in anabolic processes required for cell growth. The apparently elevated expression of InR mRNA in nervous tissue during mid- and late-embryogenesis coincides with a period of active neurite outgrowth and suggests that dIRH may be involved in this process (Garofalo, 1988).

A monoclonal antibody (Mab E1C) has been generated that recognizes the differentiated nervous system in Drosophila embryos. At the cellular blastoderm stage, Mab E1C behaves as a general ectodermal marker but, in subsequent stages, it also labels the mesoderm. As neurogenesis takes place, staining increases within the neuromeres and is almost exclusively restricted to the nervous tissue by the time neuronal differentiation is completed. In third instar larvae, Mab E1C stains the central nervous system as well as the imaginal discs, which display a staining pattern related to their degree of neuronal differentiation. No labelling can be detected in adult brains or ovaries. Western blots are consistent with this developmental profile and allow the characterization of a major glycoprotein of 135 X 10(3) Mr (135K) that cosediments with a membrane fraction prepared from embryos. Additional glycoproteins (100K and 80K) are extracted from embryo homogenates by immunoaffinity procedures. In larvae, the 100K polypeptide is not detected. The properties of the 135K and 100K components are highly reminiscent of the molecular pattern of the Drosophila insulin receptor homologue. It has been shown that a Mab directed against the human insulin receptor stains the same cells as Mab E1C in imaginal discs and in the CNS. Moreover, this Mab cross-reacts with the 135K and 100K components of the embryonic antigen E1C (Piovant, 1988).

Insulin and insulin-like growth factor (IGF) receptors are members of the tyrosine kinase family of receptors, and are thought to play an important role in the development and differentiation of neurons. The presence of an insulin-like peptide and an insulin receptor (InR) at the body wall neuromuscular junction of developing Drosophila larvae is reported. InR-like immunoreactivity is found in all body wall muscles at the motor nerve branching regions, where it surrounds synaptic boutons. The identity of this immunoreactivity as an InR was confirmed by two additional schemes: in vivo binding of labeled insulin and immunolocalization of phosphotyrosine. Both methods produce staining patterns markedly similar to InR-like immunoreactivity. The presence of an InR in whole larvae was also shown by receptor binding assays. This receptor is more specific for insulin (> 25-fold) than for IGF II, and does not appear to bind IGF I. Among the 30 muscle fibers per hemisegment, insulin-like immunoreactivity is found only on one fiber, and is localized to a subset of morphologically distinct synaptic boutons. Staining in the CNS is limited to several cell bodies in the brain lobes and in a segmental pattern throughout most of the abdominal ganglia, as well as in varicosities along the neuropil areas of the ventral ganglion and brain lobes. Insulin-like peptide and InR are first detected by early larval development, well after neuromuscular transmission begins (Gorczyca, 1993).

The isolation of the Drosophila insulin receptor gene and the recent analysis of loss of function mutants have clearly implicated insulin signaling in embryonic nervous system development. The presence of insulin in the embryo is studied and cellular processes affected by insulin in embryonic neural cells are characterized. A fraction of the cells (7.5%) in the 15-18 h Drosophila embryo contain insulin immunoreactivity. In the embryonic-derived cell line Schneider 1, human insulin is capable of stimulating proliferation and neural differentiation. Thus, the action of insulin on the developing Drosophila nervous system appears to be as pleiotropic as in vertebrates (Pimentel, 1996).

Adult flies contain a specific, high-affinity insulin-binding protein. Insulin-dependent protein tyrosine kinase activity has now been identified in Drosophila. Activity first appears at 6-12 h of embryogenesis, increases during the 12-18-h period and falls to low levels in the adult. 125I-insulin has been cross-linked specifically and with high affinity to a protein (Mr = 135,000) throughout embryogenesis and in the adult. However, during the 6-12- and 12-18-h periods of embryogenesis when insulin-dependent protein tyrosine kinase activity is expressed, another protein (Mr = 100,000) becomes cross-linked to 125I-insulin. Crosslinking to both proteins is competitively inhibited by the addition of 100 nM insulin. It is concluded that the insulin-binding and insulin-dependent protein tyrosine kinase activities of the mammalian insulin receptor are conserved in Drosophila. However, the insulin-dependent protein tyrosine kinase activity of the receptor is detected only during specific times in embryogenesis (Petruzzelli, 1985).

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).

Dicer-1-dependent Dacapo suppression acts downstream of Insulin receptor in regulating cell division of Drosophila germline stem cells

It is important to understand the regulation of stem cell division because defects in this process can cause altered tissue homeostasis or cancer. The cyclin-dependent kinase inhibitor Dacapo (Dap), a p21/p27 homolog, acts downstream of the microRNA (miRNA) pathway to regulate the cell cycle in Drosophila germline stem cells (GSCs). Tissue-extrinsic signals, including insulin, also regulate cell division of GSCs. Intrinsic and extrinsic regulators intersect in GSC division control; the Insulin receptor (InR) pathway regulates Dap levels through miRNAs, thereby controlling GSC division. Using GFP-dap 3'UTR sensors in vivo, this study shows that in GSCs the dap 3'UTR is responsive to Dicer-1, an RNA endonuclease III required for miRNA processing. Furthermore, the dap 3'UTR can be directly targeted by miR-7, miR-278 and miR-309 in luciferase assays. Consistent with this, miR-278 and miR-7 mutant GSCs are partially defective in GSC division and show abnormal cell cycle marker expression, respectively. These data suggest that the GSC cell cycle is regulated via the dap 3'UTR by multiple miRNAs. Furthermore, the GFP-dap 3'UTR sensors respond to InR but not to TGF-beta signaling, suggesting that InR signaling utilizes Dap for GSC cell cycle regulation. The miRNA-based Dap regulation may act downstream of InR signaling; Dcr-1 and Dap are required for nutrition-dependent cell cycle regulation in GSCs and reduction of dap partially rescues the cell cycle defect of InR-deficient GSCs. These data suggest that miRNA- and Dap-based cell cycle regulation in GSCs can be controlled by InR signaling (Yu, 2009).

Previous studies have shown that miRNAs may regulate Dap, thereby controlling the cell division of GSCs. This study shows that the dap 3'UTR directly responds to miRNA activities in GSCs. Using luciferase assays, miR-7, miR-278 and miR-309 were identified as miRNAs that can directly repress Dap through the dap 3'UTR in vitro. Although miR-278 and miR-7 play a role in regulating GSC division and cell cycle marker expression, respectively, neither of these mutants showed as dramatic a defect in the GSC cell cycle as Dcr-1-deficient GSCs. Thus, the dap 3'UTR may serve to integrate the effect of multiple miRNAs during cell cycle regulation. It remains possible that some miRNAs involved in this process remain to be identified. It was further shown that InR signaling controls the dap 3'UTR in GSCs. This led to an exploration of the interaction between InR signaling and miRNA/Dap cell cycle regulation. GSCs deficient for InR or Dcr-1 show similar cell cycle defects. Using starvation to control InR signaling, it was shown that both Dcr-1 and dap are required for proper InR signaling-dependent regulation of GSC division. Further, reduction of dap partially rescues the cell division defect of the InR mutant GSCs, suggesting that InR signaling regulates the cell cycle via Dap. These results suggest that miRNAs and Dap act downstream of InR signaling to regulate GSC division (Yu, 2009).

The data suggest that multiple miRNAs can regulate the 3'UTR of dap: miR-7, miR-278 and miR-309 can regulate the dap 3'UTR directly, whereas bantam and miR-8 may regulate it indirectly, or through cryptic MREs in the dap 3'UTR. Using GFP sensor assays, it was also shown that the dap 3'UTR may be directly regulated by miRNAs in the GSCs in vivo. However, which specific miRNAs control endogenous Dap levels in Drosophila GSCs remains unknown. Mammalian p21cip1 has also been shown to be a direct target for specific miRNAs of the miR-106 family, including miR-290s and miR-372. Further, the mouse miR-290 family has recently been identified as regulating the G1-S transition. In addition, miR-221 and miR-222 have been shown to regulate p27kip1, thereby promoting cell division in different mammalian cancer cell lines. Neither the miR-290 nor miR-220 family is conserved in Drosophila. Together, these results indicate that the CKIs (Dap) might be a common target for miRNAs in regulating the cell cycle in stem cells. However, the specific miRNAs that regulate the CKIs might vary between organisms (Yu, 2009).

This study reveals novel regulatory roles for miR-7 and miR-278 in the GSC cell cycle. miR-7 and miR-278 can directly target Dap. GSCs deficient for miR-278 show a mild but significant reduction in cell proliferation. Ectopic expression of miR-7 in follicle cells reduces the proportion of cells that stain positive for Dap. Furthermore, ablation of miR-7 in GSCs results in a perturbation of the frequency of CycE-positive GSCs. However, the cell division kinetics of miR-7 mutant GSCs is not reduced, by contrast with the dramatic reduction of cell division in Dcr-1-deficient GSCs. It is plausible that miR-7 and miR-278 act in concert with other miRNAs to regulate the level of Dap in GSCs and thereby contribute to cell cycle control in GSCs (Yu, 2009).

The interaction of multiple miRNAs with the dap 3'UTR might integrate information from multiple pathways. Further studies will reveal what regulates miR-7 and miR-278 expression in GSCs and which other miRNAs might act together in Dap regulation. It is known that miR-7 and the transcriptional repressor Yan mutually repress one another in the eye imaginal disk. In this model, Yan prevents transcription of miR-7 until Erk in the Egfr pathway downregulates Yan activity by phosphorylation, thereby permitting expression of miR-7. Conversely, miR-7 can repress the translation of Yan. Thus, a single pulse of Egfr signaling results in stable expression of miR-7 and repression of Yan. Whether similar regulation will be observed between miR-7 and the signaling pathways that regulate GSC division remains to be seen. It has been suggested that miR-7 might regulate downstream targets of Notch, such as Enhancer of split and Bearded. Thus, miR-7 may have a mild repressive effect on multiple targets in GSCs. Further experiments might illuminate this possibility (Yu, 2009).

miR-278, on the other hand, has been implicated in tissue growth and InR signaling. Overexpression of miR-278 promotes tissue growth in eye and wing imaginal discs. Deficiency of miR-278 leads to a reduced fat body, which is similar to the effect of impaired InR signaling in adipose tissue. Interestingly, miR-278 mutants have elevated insulin/Dilp production and a reduction of insulin sensitivity. Furthermore, miR-278 regulates expanded, which may modulate growth factor signaling including InR. Since InR signaling plays important roles in tissue growth and cell cycle control, it will be interesting to further test how miR-278 may regulate InR signaling, and whether InR signaling might regulate miR-278 in a feedback loop in GSCs (Yu, 2009).

Other miRNAs or miRNA-dependent mechanisms might also play roles in Drosophila GSCs. For example, the miRNA bantam is required for GSC maintenance. A recent study has shown that the Trim-NHL-containing protein Mei-P26, which belongs to the same family as Brain tumor (Brat), affects bantam levels and restricts cell growth and proliferation in the GSC lineage (Neumuller, 2008). Interestingly, most miRNAs are upregulated in mei-P26 mutant flies. By contrast, overexpression mei-P26 in bag of marbles (bam) mutants broadly reduces miRNA levels. This suggests that Mei-P26 regulates proliferation and maintenance of GSC lineages via miRNA levels. Since InR signaling cell-autonomously regulates GSC division but not maintenance, the possible interaction between Mei-P26 and InR signaling might be complex (Yu, 2009).

The systemic compensatory effect of insulin secretion in mammals with defective InR signaling is well documented. Insulin levels in mice with liver-specific InR (Insr - Mouse Genome Informatics) knockout are ~20-fold higher than those of control animals owing to the compensatory response of the pancreatic β-cells and impairment of insulin clearance by the liver. Knockout of the neuronal InR also leads to a mild hyperinsulinemia, indicating whole-body insulin resistance. Furthermore, the knockout of components in the InR signaling pathway, such as Akt2 and the regulatory and catalytic subunits of PI3 kinase, also leads to hyperinsulinemia and glucose intolerance. Therefore, a systemic decrease in InR signaling may lead to compensatory responses (Yu, 2009).

To understand the roles of InR signaling in the GSCs while avoiding any systemic compensatory effect the phenotypes of GSC clones were analyzed. Using a panel of cell cycle markers, it was found that InR mutant GSCs show cell cycle defects similar to those of Dcr-1 mutant GSCs: a reduction of cell division rate, an increased frequency of cells staining positive for Dap and CycE, and a decreased frequency of cells staining positive for CycB. Using GFP-dap 3'UTR sensors, it was shown that the dap 3'UTR responds to InR signaling in GSCs, suggesting that InR signaling can regulate Dap expression through the dap 3'UTR. This, together with genetic data indicating that InR/starvation-dependent cell cycle regulation requires Dcr-1 and dap, has led to the proposalthat InR signaling regulates the cell cycle through miRNAs that further regulate Dap levels. Since a reduction in dap only partially rescues the cell cycle defects of InR mutant GSCs, it is possible that InR signaling might also regulate GSC division by additional mechanisms (Yu, 2009).

InR signaling regulates the cell cycle through multiple mechanisms, mainly through the G1-S, but also partly through the G2-M, transition. Recent work has shown a delay in the G2-M transition in GSCs during C. elegans dauer formation. Starvation and InR deficiency may also affect the G2-M checkpoint in Drosophila GSCs (Hsu, 2008). This study has dissected one possible molecular pathway that InR signaling utilizes to regulate the Drosophila GSC G1-S transition and show that InR signaling can control the cell cycle through miRNA-based regulation of Dap (Yu, 2009).

Many studies have connected InR and CKIs to Tor (Target of rapamycin) or Foxo pathways downstream of InR signaling. In S. cerevisiae, the yeast homolog of p21/p27 is upregulated when Tor signaling is inhibited. Foxo, a transcription factor that can be repressed by InR signaling, is known to play important roles in nutrition-dependent cell cycle regulation by upregulating p21 and p27. In C. elegans, starvation causes L1 cell cycle arrest mediated by InR (daf-2) and Foxo (daf-16): InR represses the function of Foxo, thereby downregulating the CKI (cki-1) and upregulating the miRNA lin-4. This study has shown that a miRNA-based regulation of Dap can be coordinated by InR in Drosophila GSCs (Yu, 2009).

Insulin and insulin-like growth factors (Igf1 and Igf2) are known to play important roles in regulating metabolic and developmental processes in many stem cells. In mammals, Igf signaling is required by different stem cell types, including human and mouse ES cells for survival and self-renewal, neural stem cells for expediting the G1-S transition and cell cycle re-entry, and skeletal muscle satellite cells for promoting the G1-S transition via p27kip1 downregulation. This study has dissected the molecular mechanism of the InR pathway in another adult stem cell type, Drosophila GSCs, showing that InR signaling can regulate stem cell division through miRNA-based downregulation of the G1-S inhibitor Dap. Further studies will reveal whether miRNAs also mediate InR signaling in other stem cell types (Yu, 2009).

Insulin and Target of rapamycin signaling orchestrate the development of ovarian niche-stem cell units in Drosophila

Tissue-specific stem cells and their niches are organized into functional units that respond to external cues in order to maintain organ homeostasis. Insulin and Target of rapamycin (Tor) signaling mediate external cues that control adult niches and stem cells. Whether these pathways play a role in the establishment of niche-stem cell units during organogenesis has been little explored. This study shows that during larval development both Insulin-like receptor (InR) and or participate in the establishment of ovarian niches and germline stem cells (GSCs) in Drosophila. Tor and InR are required cell-autonomously for the proliferation of precursors for both somatic niches and GSCs. These pathways also promote the formation of terminal filaments (part of the somatic niche). Significantly, InR, but not Tor, signaling non-autonomously promotes primordial germ cell (PGC) differentiation. Somatic attenuation of the pathway retards PGC differentiation, whereas its activation results in their precocious differentiation. It was also shown that InR-mediated PGC differentiation is independent of somatic ecdysone signaling, but that further differentiation into cysts requires an ecdysone input. These results demonstrate that Tor and InR signaling actively participate in the formation of ovarian niches and stem cells by affecting both cell numbers and differentiation. The dual influence of Tor and InR on both somatic cells and PGCs ensures that these two cell populations develop coordinately. This work further identifies a novel step in the regulation of germ cell differentiation by demonstrating that following bag of marbles expression, cyst formation requires an additional hormonal input (Gancz, 2013).

Cell growth and proliferation in the larva require energy and metabolites. Accordingly, these processes are controlled by the InR and Tor pathways, which are sensors of the metabolic state of the organism. It was previously demonstrated that Insulin and Tor signaling promote the proliferation of germline precursors in C. elegans. This finding has been extended and it was shown that, in Drosophila, both somatic cells and PGCs require Tor and InR signaling cell-autonomously for their proliferation. This response is not limited to the larval growth period. The ovary is an active organ that maintains growing populations of cells. Accordingly, in the adult, somatic follicle cells, GSCs and germline cysts respond to nutrition by changing their proliferation rate. The cell-autonomous response of both soma and germline to InR and Tor signaling represents one mechanism by which the coordination of growth within an organ is achieved (Gancz, 2013).

In addition, this study found that the Tor and InR pathways affect PGC proliferation non-cell-autonomously. Smaller somatic ovaries correlate with reduced PGC proliferation, while overexpression of InR diverts PGCs from a proliferation to a differentiation program. Thus, coordination between somatic growth and germline division is monitored and corrected by more than one mechanism. It is as yet unclear how the state of somatic growth is communicated to the germline. The secondary signal might be a local ligand or might involve direct contact with the ICs. It was previously shown that the somatic cells of the ovary can control PGC proliferation via a feedback loop involving EGFR signaling in somatic cells and an unidentified signal that represses PGC proliferation. InR and Tor signaling might somehow affect this unknown signal (Gancz, 2013).

InR and Tor signaling are required for the differentiation of somatic intermingled cells (ICs) and terminal filament (TFs). ICs fail to integrate with PGCs when somatic cells have reduced InR or Tor signaling, suggesting these pathways affect IC behavior. Similarly, ovaries with reduced somatic InR and Tor signaling develop fewer TFs. This is consistent with previous observations that diet restriction (yeast deprivation) during the third instar results in reduced ovariole number. One explanation for this reduction is the reduction in TF precursors due to early proliferation defects. However, the strong reduction in TF numbers in chico-deficient ovaries, despite the relatively normal gonad size, suggests a specific role of InR in TF cell determination. Although InR signaling has been mostly associated with cell proliferation, a role for this pathway in neuronal cell differentiation has been described. The ovary might be another organ in which InR signaling affects cell differentiation. In the ovary, InR signaling can increase the number of cap cells by modulating Notch signaling, which is required for the establishment of this cell type. Thus, InR signaling acts at least twice in niche formation: first, it is required for TF formation, and then for cap cell establishment and maintenance (Gancz, 2013).

Activation of InR signaling in the soma initiates PGC differentiation precociously, whereas its repression postpones PGC differentiation. Combined, these results show that InR signaling is required for the maturation of the two components of the stem cell unit: the somatic niches and the PGCs that will occupy them. This coordination might be important at times when nutrient availability is limited, and niche formation is retarded. If PGCs differentiated normally, prior to the formation of protective niches, this would have resulted in full germ cell differentiation and lack of GSCs. Retarding PGC differentiation at times of limited nutrient availability allows additional time for niche formation prior to full depletion of the stem cell precursors (Gancz, 2013).

In InR-overexpressing ovaries, PGCs initiate their differentiation and express bam as early as the beginning of third instar (72 hours AEL). They then arrest their development for nearly 2 days; germline cysts form only following the normal elevation in ecdysone signaling, and fail to do so in its absence. One possibility is that somatic InR signaling is required (non-autonomously) for the initiation of bam transcription, while ecdysone initiates Bam translation, thereby transforming bam-expressing PGCs into proper cystoblasts. Alternatively, somatic InR might be sufficient for cystoblast formation, but further differentiation into germline cysts requires ecdysone signaling. This issue could not be resolved using the available anti-Bam antibody because this low-affinity reagent cannot recognize the naturally low levels of Bam protein in cystoblasts. It has previously been shown that PGCs can form cysts as early as second instar following hs-bam expression. Therefore, the requirement for somatic ecdysone signaling can be overridden by ectopic, high Bam expression (Gancz, 2013).

Irrespective of the mechanism by which somatic InR promotes PGC differentiation, the results suggest that the passage from a bam-expressing cell to a germline cyst might not be as direct as previously thought. Classical studies suggested that the major event in GSC differentiation is bam expression, and that Bam is both necessary and sufficient for GSCs to differentiate into germline cysts. However, these experiments were performed in ovaries in which the soma was WT. The current data suggest that the second signal required for cyst formation emanates from the soma. In support of this notion, somatic expression of a dominant-negative form of the Rho GTPase in adult germaria results in loss of contact between GSC daughter cells and escort cells. As a result, cystoblasts fail to differentiate into cysts and linger in the germarium. Thus, the second signal that emanates from the soma is required not only for PGC differentiation, but also continuously during adult oogenesis (Gancz, 2013).

Two hormonal pathways are required to promote PGC differentiation and the initiation of oogenesis: the ecdysone and the Insulin pathways. Both are required for proper somatic proliferation and lineage differentiation, and both act non-cell-autonomously to promote PGC differentiation. Epistasis analysis shows that both InR and ecdysone are required independently in the somatic ovary for bam expression in PGCs, and that ecdysone is additionally required to prepare the soma for its role in promoting cyst development (Gancz, 2013).

Of note, no direct link was detected between the ecdysone and InR pathways in the somatic cells of the ovary, suggesting that they act in parallel. However, the two pathways are linked systemically. In particular, Insulin and Tor signaling are required in the prothoracic gland for ecdysone synthesis. Because the timing of ecdysone release is intimately connected to the timing of niche and PGC differentiation, nutrition affects gonadogenesis in a systemic manner. Combined, these data suggest that InR signaling affects the ovarian stem cell precursors on multiple levels: cell-autonomously, non-cell-autonomously from the somatic ovarian cells, and systemically (Gancz, 2013).

Kao, S. H., Tseng, C. Y., Wan, C. L., Su, Y. H., Hsieh, C. C., Pi, H. and Hsu, H. J. (2014). Aging and insulin signaling differentially control normal and tumorous germline stem cells. Aging Cell 14(1): 25-34. PubMed ID: 25470527

Aging and insulin signaling differentially control normal and tumorous germline stem cells

Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. This study reports that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age-dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, GSCs from mated and unmated females behave similarly, indicating that female GSC-male communication does not affect GSCs with age. These results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging (Kao, 2014).

Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila

Drosophila larval skeletal muscles are single, multinucleated cells of different sizes that undergo tremendous growth within a few days. The mechanisms underlying this growth in concert with overall body growth are unknown. The size of individual muscles correlates with the number of nuclei per muscle cell and with increasing nuclear ploidy during development. Inhibition of Insulin receptor (InR; Insulin-like receptor) signaling in muscles autonomously reduces muscle size and systemically affects the size of other tissues, organs and indeed the entire body, most likely by regulating feeding behavior. In muscles, InR/Tor signaling, Foxo and dMyc (Diminutive) are key regulators of endoreplication, which is necessary but not sufficient to induce growth. Mechanistically, InR/Foxo signaling controls cell cycle progression by modulating dmyc expression and dMyc transcriptional activity. Thus, maximal dMyc transcriptional activity depends on InR to control muscle mass, which in turn induces a systemic behavioral response to allocate body size and proportions (Demontis, 2009).

Therefore, interplay between InR/Tor signaling, Foxo and dMyc activity regulates muscle growth that occurs during Drosophila larval development, in part via the induction of endoreplication. Interestingly, the extent of muscle growth is sensed systemically, regulates feeding behavior and, in turn, influences the size of other tissues and indeed the whole body. Thus, the growth of a single tissue is sensed systemically via modulating a whole-organism behavior (Demontis, 2009).

dMyc, as well as activation of InR signaling, can promote endoreplication in muscles, whereas Foxo and inhibitors of dMyc and of InR/Tor have the opposite effect. dMyc is likely to regulate the expression of genes required for multiple G-S and S-G transitions during endoreplication, similar to vertebrate Myc, which regulates key cell-cycle regulators including cyclin D2, cyclin E, and the cyclin kinase inhibitors p21 and p27 (Cdkn1a and Cdkn1b, respectively). Indeed, aberrant levels of Cyclin E block muscle growth, indicating that proper muscle growth requires tight control of the expression and activity of endoreplication genes. Further, endoreplication is also modulated by Foxo, which is activated in conditions of nutrient starvation, impaired InR/Tor signaling and by other cell stressors. Foxo presumably regulates cell cycle progression at least in part by modulating the expression of evolutionarily conserved Foxo/Myc-target genes, such as dacapo (the Drosophila p21/p27 homolog) and Cyclin E, that regulate the G1-S transition. Interestingly, Foxo and Myc might control different steps in the activation of common target genes (Demontis, 2009).

In addition, it was found that active Foxo can also inhibit dMyc protein activity and regulates dmyc gene expression. Mechanistically, Foxo could influence dMyc activity in several ways. First, it might physically interact with dMyc, although no evidence was found to support this notion. Second, Foxo could regulate the expression of genes that target dMyc for proteasomal degradation, including several ubiquitin E3 ligases that are induced by Foxo during muscle atrophy in mice and humans. However, by analyzing dMyc protein levels by western blot, no significant dMyc protein instability was found upon Foxo overexpression. Third, Foxo might promote the expression of transcriptional regulators that oppose dMyc function, including Mad/Mnt, although no substantial increase in dmnt mRNA levels was detected upon Foxo activation in muscles. Possibly, the expression of other dMyc regulators might be affected by Foxo. Future experiments will be needed to dissect the Foxo-dMyc interaction (Demontis, 2009).

Finally, by manipulating muscle growth and/or endoreplication, it was found that in muscles the ratio of cell size to nuclear size is not constant, and increased nuclear size and DNA content, indicative of ploidy, is necessary but not sufficient to drive growth. Usually, an increase in cell size is matched by an increase in nuclear size, which commonly parallels increases in nuclear ploidy. However, the current findings indicate that in muscles, dMyc-driven variation in nuclear size and ploidy is permissive but not sufficient for substantial growth, even in the presence of increased biogenesis of nucleoli and expression of genes involved in protein translation. This is different from fat body cells, in which dmyc overexpression induces endoreplication and proportional cell growth. Thus, additional instructive signals, possibly modulating protein synthesis, mitochondriogenesis, ribosome biogenesis, sarcomere assembly, and other anabolic responses must be concomitantly received to promote maximal muscle growth. Therefore, increases in cell size and nuclear ploidy are surprisingly uncoupled during muscle growth (Demontis, 2009).

Little is known about the mechanisms that control and coordinate cell, organ and body size, and in particular how muscle growth is matched with the growth of other tissues and of the entire organism. Inhibition of InR/Tor signaling and dMyc activity in muscles impairs, in addition to muscle mass, the size of the entire body and of other internal organs. Similarly, overexpression of Cyclin E in muscles also results in autonomous and systemic growth defects, indicating that, at least in some cases, modulation of muscle growth by means independent from InR signaling can be sensed systemically. In the larva, endoreplicating tissues and organs (gut, salivary glands, epidermis, fat body) are severely affected, whereas non-endoreplicating tissues (brain and imaginal discs) are less affected, indicating distinct tissue responsiveness to this regulation. Similarly, inhibition of Tor signaling in the fat body also primarily affects the size of endoreplicating tissues (Demontis, 2009).

Non-autonomous regulation of tissue size may rely on humoral factors (e.g. hormone-binding proteins, hormones, metabolites) produced by muscles in response to achieving a certain mass. However, alternative models are possible. In particular, decreased and increased larval feeding, respectively, were observed upon inhibition and activation of InR signaling in muscles. This whole-organism behavioral adaptation is possibly due to decreased and increased efficiency of smaller and bigger muscles, respectively, and to regulated expression of neuropeptides that hormonally control feeding behavior. As a consequence of the regulation of feeding behavior, nutrient uptake is decreased and larval growth is blocked in the cells of endoreplicating tissues, which are extremely sensitive to poor nutritional conditions, and to a lesser extent in non-endoreplicating tissues, which are more resistant to limited nutritional supply. In turn, increased or decreased size of non-muscle tissues arise as a consequence of abnormal feeding. Thus, muscle size coordinates with the size of other organs and of the entire body, at least in part via a systemic, behavioral response. Distinct tissues are differently sensitive to this regulation, resulting in altered body proportions (Demontis, 2009).

Understanding the mechanisms regulating muscle mass is of special interest because they underline the etiology of several human diseases. Directly relevant to these studies, both MYC and InR (INSR) signaling have been found to regulate muscle growth and maintenance in humans. Further, muscle atrophy is triggered by FOXO activation in several pathological conditions. In addition, MYC function has been implicated in heart hypertrophy, a process that is conversely regulated by FOXO (Demontis, 2009).

The findings that Foxo functionally antagonizes dMyc during the growth of Drosophila muscles suggest that these factors might also interact similarly in humans. Consistent with this hypothesis, FOXO and MYC regulate, in opposite fashions, the atrophic and hypertrophic programs in human skeletal muscles and cardiomyocytes, and display complementary gene expression and activity in these contexts (Demontis, 2009).

Finally, the finding that during larval development, inhibition of InR signaling in muscles has profound systemic effects might also reflect physiological conditions found in humans. Indeed, defective responsiveness of muscles to Insulin during type II diabetes has autonomous effects on muscle maintenance that are associated with systemic effects on the metabolism of the entire organism, contributing to the improper control of glycemia and the development of metabolic syndrome. This study has identified feeding behavior as part of the systemic response that in Drosophila senses perturbations in muscle mass. These findings might help further elucidate the signals involved in metabolic and growth homeostasis, which may be conserved across evolution (Demontis, 2009).

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 receptor-mediated signaling via phospholipase C-γ regulates growth and differentiation in Drosophila.

Coordination between growth and patterning/differentiation is critical if appropriate final organ structure and size is to be achieved. Understanding how these two processes are regulated is therefore a fundamental and as yet incompletely answered question. This study shows through genetic analysis that the phospholipase C-γ (PLC-γ) encoded by small wing (sl) acts as such a link between growth and patterning/differentiation by modulating some MAPK outputs once activated by the insulin pathway; particularly, sl promotes growth and suppresses ectopic differentiation in the developing eye and wing, allowing cells to attain a normal size and differentiate properly. sl mutants have previously been shown to have a combination of both growth and patterning/differentiation phenotypes: small wings, ectopic wing veins, and extra R7 photoreceptor cells. This study shows that PLC-γ activated by the insulin pathway participates broadly and positively during cell growth modulating EGF pathway activity, whereas in cell differentiation PLC-γ activated by the insulin receptor negatively regulates the EGF pathway. These roles require different SH2 domains of PLC-γ, and act via classic PLC-γ signaling and EGF ligand processing. By means of PLC-γ, the insulin receptor therefore modulates differentiation as well as growth. Overall, these results provide evidence that PLC-γ acts during development at a time when growth ends and differentiation begins, and is important for proper coordination of these two processes (Murillo-Maldonado, 2011).

By measuring cell density, this study shows that sl mutant wings have a reduction in cell growth but not cell proliferation. This defect is qualitatively similar to mutations in MAPK signaling; cells with homozygous mutations for members of this pathway have higher cell densities, suggesting smaller cells. Of the several signaling pathways known to be involved in Drosophila wing growth, only the MAPK and insulin pathways are triggered by tyrosine kinase receptors that are likely to activate Sl. The results show that indeed both pathways are genetically linked to Sl in promoting cell growth, probably acting in a concerted fashion; further molecular studies will be required to reveal the molecular mechanisms and physical interactions that allow this link. Sl signaling thus provides a means for coordinating growth by forming a regulatory link between the MAPK and insulin pathways. In this scenario, Sl activated by the insulin pathway would function by modulating MAPK output; that is to say, to reduce somewhat the levels of MAPK activity, but not to stop it, as no MAPK activity leads to no growth and cell death, and too much MAPK activity leads to ectopic differentiation and reduced growth (Murillo-Maldonado, 2011).

Sl regulates cellular growth in the eye. Whole eyes are smaller, and the difference in size can be largely explained by the presence of fewer ommatidia. This means that sl mutant eyes very likely contain fewer cells, despite the fact that some ommatidia sport one or two extra R7 cells, as the number of cells missing due to reduced numbers of ommatidia is bigger than the number of extra R7 cells present. This suggests either reduced proliferation or increased cell death in differentiating sl mutant eyes, and is different from the growth defect found in wings, yet consistent with a moderate requirement of MAPK output to promote growth and cellular survival (Murillo-Maldonado, 2011).

Not only is cell size reduced to a similar extent in both the eye and wing of sl homozygotes; the adult animal as a whole has reduced mass. Given that the reduction in mass (8%) is of a similar magnitude to the reduction in cell size in the eye (15%) and wing (20%), the most parsimonious explanation for this change in mass is that the same Sl functions found in the eye and wing are required more generally throughout the animal, suggesting that cell size may be reduced in many tissues. However, it was found that the reduced growth observed in the adult was not reflected by a reduction in length of sl mutant pupae. This is in contrast to mutations of other genes involved in growth control, such as the neurofibromin 1 gene, which shows a significant reduction in pupal length. This might be because sl has a relatively small effect on growth, varying between 5% and 20% in different contexts, so this sample may not have been large enough to observe a small change in mean length. Given that Sl does not appear to affect the length of appendages other than the wing, it may be that there are other compensatory effects resulting from lost Sl function that maintain the pupal case at an approximately wild-type length (Murillo-Maldonado, 2011).

Another complementary explanation for the reduction in adult mass is via a role for Sl on nutrient sensing. As Sl is clearly involved in insulin signaling, and as insulin is required for integrating nutrient sensation in Drosophila, the effect on mass might be a combination of impacts on both growth signaling and nutrient sensing (Murillo-Maldonado, 2011).

It is proposed that the overall role for Sl is to act as a pro-growth agent, allowing cells and tissues to attain normal numbers and sizes. This is achieved by dampening MAPK output in growth control in a non-cell autonomous manner, by restricting processing of EGFR ligand(s), as shown previously for R7 cell differentiation. Since both the MAPK and insulin pathways initially act to favor proliferation and growth, it is proposed that Sl functions here under insulin pathway control, allowing growth to continue, preventing ectopic differentiation. There are several ways in which it could do so: by directing activated MAPK to a different cellular compartment (cytosolic versus nuclear or by controlling overall strength and duration of signaling, examples of which have been shown to elicit such changes in developing wing cells in both Drosophila and PC12 cells (Murillo-Maldonado, 2011).

A central function of all phospholipase C enzymes is hydrolysis of PIP2. In this study has shown that regulation of growth and differentiation by Sl must depend on PIP2 hydrolysis to some extent, because of the interaction between sl and mutations in IP3R, PKC53E and Rack1. Also, by means of genetic tests, it was found that Sl requires the Spi processing machinery (S, Rho) to regulate growth and differentiation. It has previously been shown that Sl acts on Spi processing during R7 differentiation, by favoring Spi retention in the endoplasmic reticulum. In order to rationalize Sl function in all the phenotypes studied, it was reasoned that by inhibiting Spitz processing, Sl could delay initiation of differentiation, allowing still undifferentiated cells to grow and attain a normal size before the onset of differentiation. Sl modes of action in growth and differentiation may be different; sl alleles affecting the wing but not the eye is strong evidence for this assertion (Murillo-Maldonado, 2011).

In general, during growth, Sl activated by the insulin pathway acts as a liaison regulating MAPK pathway ligand processing, to promote MAPK activation to a level permitting growth. In agreement with a well-characterized case in mammalian cells, it is proposed that this level of activity of MAPK is different from the level required for differentiation; either it is of a different duration, or of an overall different stimulation level, or happening at a different time. Alternatively it occurs in a different subcellular compartment from that required for differentiation, acting thru Sl regulation of Spi processing. This scenario also requires both the MAPK and the insulin pathways to be active for cellular growth. Conversely, for differentiation, reduced insulin receptor signaling leads to altered (lower) levels of Sl activation and augmented Spi processing, and this in turn allows MAPK activation in a manner consistent with promotion of differentiation. This could either be caused by longer or stronger MAPK stimulation, as documented for PC12 cells, since lower Sl activity now allows higher levels of MAPK ligand processing, and/or by compartmentalization of the activated MAPK pathway, as shown for the Drosophila wing, besides happening at different times during development. In this second case, only the MAPK pathway is required to be fully active. Finally, loss-of-function mutant conditions for sl lead to ectopic differentiation at the expense of growth (Murillo-Maldonado, 2011).

Taken together, these results indicate that Sl participates in fine coordination of growth and differentiation during development. Although Sl is not essential for wing or eye growth and development, it is necessary to achieve appropriate final structure and size. In the absence of Sl function, these tissues arrest growth prematurely and probably initiate differentiation earlier, resulting in ectopic differentiation while attaining smaller cellular sizes. As such, Sl can be seen as exerting a kind of 'parental control' that protects cells from differentiating before attaining a normal size. This function requires Sl to change cellular behavior from growth (or possibly inhibition of differentiation) to differentiation in a short period of time (Murillo-Maldonado, 2011).

PLC-γ1 has been demonstrated to be a phosphorylation target of MAPK, and some PKC isoforms can phosphorylate PLC-γ without affecting PIP2 hydrolysis so it is clear that there is a complex interplay of signaling among this set of molecules following RTK activation. Further study of the dynamics of Sl-regulated EGF/MAPK signaling in space and time during wing and eye development in Drosophila may help to expose more of this network (Murillo-Maldonado, 2011).

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

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

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

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

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

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

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

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

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

Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain

Glial cells are essential for the development and function of the nervous system. In the mammalian brain, vast numbers of glia of several different functional types are generated during late embryonic and early fetal development. However, the molecular cues that instruct gliogenesis and determine glial cell type are poorly understood. During post-embryonic development, the number of glia in the Drosophila larval brain increases dramatically, potentially providing a powerful model for understanding gliogenesis. Using glial-specific clonal analysis this study found that perineural glia and cortex glia proliferate extensively through symmetric cell division in the post-embryonic brain. Using pan-glial inhibition and loss-of-function clonal analysis it was found that Insulin-like receptor (InR)/Target of rapamycin (TOR) signalling is required for the proliferation of perineural glia. Fibroblast growth factor (FGF) signalling is also required for perineural glia proliferation and acts synergistically with the InR/TOR pathway. Cortex glia require InR in part, but not downstream components of the TOR pathway, for proliferation. Moreover, cortex glia absolutely require FGF signalling, such that inhibition of the FGF pathway almost completely blocks the generation of cortex glia. Neuronal expression of the FGF receptor ligand Pyramus is also required for the generation of cortex glia, suggesting a mechanism whereby neuronal FGF expression coordinates neurogenesis and cortex gliogenesis. In summary, this study has identified two major pathways that control perineural and cortex gliogenesis in the post-embryonic brain and has shown that the molecular circuitry required is lineage specific (Avet-Rochex, 2012).

The correct control of gliogenesis is crucial to CNS development and the Drosophila post-embryonic nervous system is a powerful model for elucidating the molecular players that control this process. This study has identified two separate glial populations that proliferate extensively and have defined the key molecular players that control their genesis and proliferation. Perineural and cortex glia both use insulin and FGF signalling in a concerted manner, but the requirements for these pathways are different in each glial type. The data suggest a model that describes the molecular requirements for post-embryonic gliogenesis in each of these glial types in the brain (Avet-Rochex, 2012).

The results show that Pyramus is expressed by perineural glia to activate FGF signalling in adjacent glia and acts in parallel to InR/TOR signalling (activated by the expression of Dilp6). These two pathways act synergistically to generate the correct complement of perineural glia. The results also show that cortex glia proliferation is controlled by FGF signalling through FGFR (Htl) and the Ras/MAPK pathway. Pyr expression is required from both glia and neurons and acts non-cell-autonomously. Neuronal Pyr expression activates the FGFR on adjacent cortex glia, thereby coordinating neurogenesis and glial proliferation. InR is also partially required in cortex glia and is likely to signal through the Ras/MAPK pathway (Avet-Rochex, 2012).

Using both pan-glial inhibition and LOF clonal analysis this study has shown that the InR/TOR pathway is required for perineural glia proliferation. InR/TOR signalling has widespread roles in nervous system development and a role has been demonstrated for this pathway in the temporal control of neurogenesis (Bateman, 2004; McNeill, 2008). InR can be activated by any one of seven DILPs encoded by the Drosophila genome, which can act redundantly by compensating for each other. dilp6 is expressed in most glia during larval development, including perineural and cortex glia, and that dilp6 mutants have reduced gliogenesis. The dilp6 phenotype is weaker than that associated with the inhibition of downstream components of the InR/TOR pathway, suggesting that other DILPs might be able to compensate for the absence of dilp6 expression in glia (Gronke, 2010). Pan-glial inhibition and clonal analysis also demonstrated that the FGF pathway is required for normal levels of perineural glia proliferation. FGF signalling is activated in perineural glia by paracrine expression of Pyr. Inhibition of either the InR/TOR or FGF pathway reduced perineural glia proliferation by about half, so tests were performed to see whether these two pathways act together. The data demonstrate that inhibition of both pathways simultaneously has a synergistic effect, suggesting that these two pathways act in parallel, rather than sequentially, and that their combined activities generate the large numbers of perineural glia found in the adult brain (Avet-Rochex, 2012).

Cortex glia employ a molecular mechanism distinct from that of perineural glia to regulate their proliferation. Cortex glia have a clear requirement for InR, as InR mutant cortex clones are significantly reduced in size. The early events in post-embryonic gliogenesis are poorly understood, but FGF signalling is likely to be required during this stage as LOF clones for components of this pathway almost completely block cortex gliogenesis. These data suggest that InR acts in parallel to FGF signalling in these cells, as loss of InR combined with activation of FGF signalling only partially rescues the InR phenotype. Interestingly, the PI3K/TOR pathway is not required in cortex glia, suggesting that InR signals through the Ras/MAPK pathway to control cortex glia proliferation (Avet-Rochex, 2012).

The FGF pathway in cortex glia responds to paracrine Pyr expression from both glia and neurons. Expression from both glia and neurons is required to activate the pathway and stimulate cortex gliogenesis. Neuronal regulation of glial FGF signalling enables cortical neurogenesis to modulate the rate of gliogenesis, so that the requisite number of glia are generated to correctly enwrap and support developing cortical neurons. Recent studies have also identified a mechanism by which DILP secretion by glia controls neuroblast cell-cycle re-entry in the Drosophila early post-embryonic CNS. Thus, neurons and glia mutually regulate each other's proliferation to coordinate correct brain development (Avet-Rochex, 2012).

This study has shown that two major glial populations in the larval brain, perineural and cortex glia, are generated by glial proliferation rather than differentiation from neuroglioblast or glioblast precursors. Differentiation of most embryonic glia from neuroglioblasts in the VNC requires the transcription factor glial cells missing (gcm), which is both necessary and sufficient for glial cell fate. In the larval brain the role of gcm is much more restricted and it is not expressed in, nor required for, generation of perineural glia. Thus, the developmental constraints on gliogenesis in the embryonic and larval CNS are distinct. The larval brain undergoes a dramatic increase in size during the third instar, which might favour a proliferative mode, rather than continuous differentiation from a progenitor cell type (Avet-Rochex, 2012).

Glial dysfunction is a major contributor to human disease. The release of toxic factors from astrocytes has been suggested to be a contributory factor in amyotrophic lateral sclerosis and astrocytes might also play a role in the clearance of toxic Aβ in Alzheimer’s disease . Rett syndrome is an autism spectrum disorder caused by LOF of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Astrocytes from MeCP2-deficient mice proliferate slowly and have been suggested to cause aberrant neuronal development. This hypothesis was recently confirmed by astrocyte-specific re-expression of Mecp2 in MeCP2-deficient mice, which improved the neuronal morphology, lifespan and behavioural phenotypes associated with Rett syndrome. Characterisation of the molecular control of gliogenesis during development might lead to a better understanding of such diseases (Avet-Rochex, 2012).

The insulin receptor is required for the development of the Drosophila peripheral nervous system

The Insulin Receptor (InR) in Drosophila presents features conserved in its mammalian counterparts. InR is required for growth; it is expressed in the central and embryonic nervous system and modulates the time of differentiation of the eye photoreceptor without altering cell fate. This study shows that the InR is required for the formation of the peripheral nervous system during larval development and more particularly for the formation of sensory organ precursors (SOPs) on the fly notum and scutellum. SOPs arise in the proneural cluster that expresses high levels of the proneural proteins Achaete (Ac) and Scute (Sc). The other cells will become epidermis due to lateral inhibition induced by the Notch (N) receptor signal that prevents its neighbors from adopting a neural fate. In addition, misexpression of the InR or of other components of the pathway (PTEN, Akt, FOXO) induces the development of an abnormal number of macrochaetes, which are Drosophila mechanoreceptors. These data suggest that InR regulates the neural genes ac, sc and sens. The FOXO transcription factor, which becomes localized in the cytoplasm upon insulin uptake, displays strong genetic interaction with the InR and is involved in Ac regulation. The genetic interactions between the epidermal growth factor receptor (EGFR), Ras and InR/FOXO suggest that these proteins cooperate to induce neural gene expression. Moreover, InR/FOXO is probably involved in the lateral inhibition process, since genetic interactions with N are highly significant. These results show that the InR can alter cell fate, independently of its function in cell growth and proliferation (Dutrieux, 2013).

A model is proposed in which the InR receptor plays a role in the development of the peripheral nervous system mainly through FOXO cell localization independently of its role in proliferation and apoptosis. The role of the InR/FOXO pathway appears early in PNS development before SOP formation. The use of different mutants involved in growth indicates that the TOR pathway does not play a major role in the phenotypes observed. The results using genetic and molecular methods strongly suggest that InR/FOXO controls the level of proneuronal genes such as ac, sc and Sens early in PNS development. This explains the interaction observed with N55e11 (Dutrieux, 2013).

Several arguments indicate that the phenotypes observed when InR is overexpressed are not due, at least for the most part, to proliferation, growth or lack of apoptosis. First using anti-PH3 staining that allows to visualize mitotic cells, no extra mitoses are observed in the clusterOverexpression of genes such as dE2F1, or dacapo did not lead to a significant increase or decrease in the number of macrochaetes. In addition co-expression of these genes with InR indicates no interaction. Moreover, the effects of InR and FOXO when overexpressed on respectively the increase and the decrease in cell number, could be estimated by the number of Ac-positive cells in the DC and SC clusters. No significant differences were observed between the control and the overexpressed strain (either InR or FOXO) in the number of cells positive for Ac. If the possibility that proliferation is somehow involved in cluster size cannot be discarded, it does not account for the effects observed since the ratio of Sens-positive cells when InR is overexpressed over the control strain is much higher than the ratio of Ac-positive cells. A similar role for FOXO in apoptosis could also be discarded on the same basis. No clear interactions were observed between FOXO and genes involved in inhibition of apoptosis like diap1 (Dutrieux, 2013).

Along the same line it has been shown that the InR/TOR pathway plays a role in controlling the time of neural differentiation. This has been observed in photoreceptor formation but also in the chordotonal organs of the leg that develop on the same basis as thoracic bristles. The dynamic formation of the SOPs, particularly after a block of InR signaling was undertaken. No differences were observed before the end third larval instar in the test and in the overexpressed strain. Only an increase in the number of positive Sens stained cells are observed in the sca>InR strain (Dutrieux, 2013).

Using Pros staining that marks pIIb cells, this study shows that staining appears in the late third instar larvae at the level of DC SOPs in sca>InR; this is not observed in the control strain. In addition in sca>FOXO RNAi wing discs it also leads to Pros staining. This indicates that the time of differentiation is advanced in the InR strain through the absence of nuclear FOXO. However it was verified that in very early third instar larvae the first scutellar SOP appears at the same time in the control and in the overexpressed strains and that no differences were observed in mid third instar (Dutrieux, 2013).

In addition the observations show that the increase in the number of macrochaetes in sca>InR is independent of the TOR pathway since none of the members induces a similar phenotype as does InR or interacts either with InR or FOXO in this process. However, some interactions were observed with raptor and Rheb that could be the consequence for the latter of its role in PIIa and PIIb formation regulating N (Dutrieux, 2013).

Are InR and FOXO acting on the same target in SOP formation? Several arguments are in favor of this possibility. First underexpression experiments (InR clones, InR RNAi or FOXO RNAi overexpression and FOXO homozygotes and even heterozygotes,) induce exactly opposite phenotypes. This is also true for overexpression experiments with InR and hFOXO3a-TM. Moreover overexpression of both transgenes leads to an intermediate phenotype, very different from the control phenotype. Finally, overexpression of InR in a heterozygote FOXO mutant background leads to an increase in the number of macrochaetes compared to InR alone. FOXO null flies are fully viable and do not usually display any phenotype. However an increase in the number of pDC and aSC macrochaetes is observed in some FOXO homozygotes and even heterozygotes that are nor observed in the control strain. This could indicate that FOXO function is in part dispensable. Even if the InR/FOXO double heterozygote is completely normal, the double null mutant InR/FOXO shows either an excess or a lack of macrochaetes, that is in favor of the hypothesis that InR acts through FOXO. FOXO null clones do not display any phenotype comparable to FOXO RNAi overexpression. However overexpression of InR in a FOXO null clone leads to stronger phenotypes than overexpression of InR alone in a clone. Yet, it cannot be excluded that part of the InR overexpression phenotype is not due to the absence of FOXO or its cytoplasmic retention (Dutrieux, 2013).

The absence of FOXO, using FOXO RNAi, or its retention in the cytoplasm by InR or Akt overexpression produces the same neurogenic phenotypes that are exactly the opposite when nuclear hFOXO3a-TM is overexpressed. In addition overexpression of both hFOXO3a-TM and InR leads to a decrease in the number of highly positive Ac and Sens expressing cells compared to overexpression of InR alone. Finally, overexpression of FOXO RNAi in dpp regulatory sequences, induces Ac expression. All these results should be explained by the same molecular process. One possibility would be that InR/FOXO regulates one or several neural genes involved in cluster formation and maintenance. The results are in favor of the hypothesis that genes of the Ac/Sc complex could be regulated by InR. Either InR via nuclear FOXO represses the Ac/Sc pathway, or FOXO activates a repressor of the pathway (Dutrieux, 2013).

Since it has been well established that InR induces cell proliferation, it remains possible that these functions could affect the size of the proneural clusters when the genes are overexpressed. However, when the number of the Ac-positive cells in the DC and SC clusters in the different genotypes was estimated, it was not significantly different (Dutrieux, 2013).

Several relevant arguments exist suggesting that InR is necessary for SOP formation and regulation of neural gene expression. (1) The phenotype of the overexpression experiments either with InR or with InR RNAi suggests that InR perturbs the normal pattern of singling out a cell in the proneural cluster that will become an SOP. The fact that the sensitive period occurs in the late second/beginning third instar is in accordance with this hypothesis. The phenotype of the InR null clones comfort this hypothesis. (2) When InR is overexpressed the level of Ac is significantly higher. This is confirmed by the IMARIS technique that estimated that in this genotype, the number of cells with the highest scores (106 and 107 units) is larger than in the control strain. These 'highly Ac-positive cells' seem to also be Sens positive cells indicating a correlation between the two events. (3) In sca>InR the level of Sens, measured by the IMARIS technique is higher than in the test raising the possibility that InR regulates several neural genes independently. However another possibility would be that this high Sens expression level would be indirectly due to the induction by InR of a Sens-positive regulator such as sc. (4) Several sc enhancers are regulated by InR, the sc promoter, and the SRV and DC enhancers. As sc is auto-regulated through its different enhancers, it is difficult to evaluate if a specific enhancer is involved although the effect on the 3.8 kb sc promoter is the most striking. For FOXO the absence of FOXO using the FOXO RNAi strain shows that Ac is induced. The double expression of InR and hFOXO3a-TM produces an intermediate phenotype and decreases the effects of InR, on Ac and Sens expression. The results using the sc enhancers when hFOXO3a-TM is overexpressed showed that only a decrease in the expression of the SRV enhancer is observed. However, the phenotypes observed in sca>hFOXO3a-TM agree with the hypothesis of repression of ac and sc by hFOXO3a-TM. As expected, overexpression of FOXO RNAi induces sc-lacZ enhancer. (5) Overexpression of both InR and sc leads to a significant increase in the effect of a single transgene. This indicates that both transgenes have a common target; one of them could be sc itself. An opposite effect is observed with constitutively active hFOXO3a-TM. This favors the model whereby InR and FOXO act in opposite ways on the sc target in SOP formation. (6) Highly significant genetic interactions are observed between sc and InR, and sc and FOXO. (7) Another gene charlatan (chn) which is both upstream and downstream of sc, strongly interacts genetically with InR (Dutrieux, 2013).

Lateral inhibition is determined by the activity of the N receptor. When N is mutated, cell fate changes and extra macrochaete singling appear. Using the N deletion (N55e11) to test possible genetic interaction with InR and with FOXO in heterozygote females, interaction was observed with the InR RNAi strain. Moreover strong interaction is observed with InR overexpression. This indicates that InR impairs lateral inhibition and cooperates with N in this process. In parallel, as for Inr overexpression, the absence of nuclear FOXO either using FOXO25 homozygotes (or even heterozygotes) or FOXO RNAi overexpression induces an increase in the neurogenic phenotype. With this latter strain, tufted microchaetes were observed, indicating that FOXO could also act later in development. Overexpression of hFOXO3a-TM displays highly significant interaction with N55e11 as the neurogenic phenotype is increased compared to overexpression in a wild type background. However, overexpression of InR RNAi in a N55e11 heterozygote background leads to a significant increase but only at the level of aSC, raising the possibility of a local interaction or appearing at a specific time for the different clusters (Dutrieux, 2013).

Moreover the fact that there is no differences when Suppressor of Hairless (Su(H)) which transduces the N pathway, is expressed with or without the InR, indicates that lateral inhibition is not affected. In addition in the InR strain, Sens stained cells were clearly individualized and separated from one another. These results clearly indicate that InR and FOXO act with N on the choice of the cell that will become an SOP (Dutrieux, 2013).

EGFR has also been implicated in macrochaete development. Indeed EGFR mutants and EGFR null clones display macrochaete phenotypes. This could be explained since in EGFR hypomorphic mutants the level of Sc is reduced in some clusters and increased in others suggesting a different requirement of EGFR for the different SOPs. If RasV12 was overexpressed with an ubiquitous driver, sc was ectopically expressed. Thus, Ac/Sc induction by Ras overrules lateral inhibition due to N. Moreover N downregulation enhances EGFR signaling. A model has been established of antagonist interaction between EGFR and N in which Ac/Sc activates both pathways that in turn act on the same SOP specific enhancers (Dutrieux, 2013).

Moreover, the InR/TOR pathway regulates the expression of some of the components of the EGFR signaling pathway such as argos, rhomboid and pointed. The results suggest that both the InR and the EGFR/Ras pathways induce sc in a synergic manner and this further overrules the lateral inhibition mechanism induced by N. The fact that overexpression of RasV12 in an InR null heterozygote background significantly lowers the phenotype observed with RasV12 only, is in agreement with this hypothesis. The interactions observed with the EGFR RNAi strain seem to be FOXO independent (Dutrieux, 2013).

Taken together these results show that InR and several components of the pathway such as PTEN, Akt and FOXO are involved in PNS development independently of their role in growth, proliferation and delay in the time of neural differentiation. The function of InR in PNS development seems to be independent of TOR/4E-BP. FOXO cytoplasmic retention either by InR activation or by the use of FOXO RNAi produces opposite phenotypes suggesting that nuclear FOXO could be a repressor of PNS development. These results using antibody staining and reporters of sc enhancers indicate that InR targets are the neural genes ac, sc and sens. However, as most of these neural genes display a complex co-regulation, it is difficult to demonstrate whether or not sc is the primary target of the pathway. A strong interaction is observed between the EGFR/Ras pathways and InR suggesting that both could act together to induce neural gene expression and this would explain the strong interaction observed between InR/FOXO and N (Dutrieux, 2013).

Regulation of cuticle pigmentation in Drosophila by the nutrient sensing insulin and TOR signaling pathways

Insect pigmentation is a phenotypically plastic trait that plays a role in thermoregulation, desiccation tolerance, mimicry, and sexual selection. The extent and pattern of pigmentation of the abdomen and thorax in Drosophila melanogaster is affected by environmental factors such a growth temperature and access to the substrates necessary for melanin biosynthesis. This study aimed to determine the effect of nutritional status during development on adult pigmentation and test whether nutrient sensing through the Insulin/IGF and target of rapamycin (TOR) pathways regulates the melanization of adult cuticle in Drosophila. Flies reared on low quality food were shown to exhibit decreased pigmentation, which can be phenocopied by inhibiting expression of the Insulin receptor (InR) throughout the entire fly during mid to late pupation. The loss of Insulin signaling through PI3K/Akt and FOXO in the epidermis underlying the developing adult cuticle causes a similar decrease in adult pigmentation, suggesting that Insulin signaling acts in a cell autonomous manner to regulate cuticle melanization. In addition, TOR signaling increases pigmentation in a cell autonomous manner, most likely through increased S6K activity. These results suggest that nutrient sensing through the Insulin/IGF and TOR pathways couples cuticle pigmentation of both male and female Drosophila with their nutritional status during metamorphosis (Shakhamtsir, 2013).

Insulin/IGF-Regulated Size Scaling of Neuroendocrine Cells Expressing the bHLH Transcription Factor Dimmed in Drosophila

Neurons and other cells display a large variation in size in an organism. Thus, a fundamental question is how growth of individual cells and their organelles is regulated. Is size scaling of individual neurons regulated post-mitotically, independent of growth of the entire CNS? Although the role of insulin/IGF-signaling (IIS) in growth of tissues and whole organisms is well established, it is not known whether it regulates the size of individual neurons. The role of IIS in the size scaling of neurons in the Drosophila CNS was studied. By targeted genetic manipulations of insulin receptor (dInR) expression in a variety of neuron types it was demonstrated that the cell size is affected only in neuroendocrine cells specified by the bHLH transcription factor DimmedD (Dimm). Several populations of Dimm-positive neurons tested displayed enlarged cell bodies after overexpression of the dInR, as well as PI3 kinase and Akt1 (protein kinase B), whereas Dimm-negative neurons did not respond to dInR manipulations. Knockdown of these components produce the opposite phenotype. Increased growth can also be induced by targeted overexpression of nutrient-dependent TOR (target of rapamycin) signaling components, such as Rheb (small GTPase), Tor and S6K (S6 kinase). After Dimm-knockdown in neuroendocrine cells manipulations of dInR expression have significantly less effects on cell size. It was also shown that dInR expression in neuroendocrine cells can be altered by up or down-regulation of Dimm. This novel dInR-regulated size scaling is seen during postembryonic development, continues in the aging adult and is diet dependent. The increase in cell size includes cell body, axon terminations, nucleus and Golgi apparatus. It is suggested that the dInR-mediated scaling of neuroendocrine cells is part of a plasticity that adapts the secretory capacity to changing physiological conditions and nutrient-dependent organismal growth (Luo, 2013).


The insulin-binding and protein tyrosine kinase subunits of the Drosophila melanogaster insulin receptor homolog have been identified and characterized by using antipeptide antibodies elicited to the deduced amino acid sequence of the alpha and beta subunits of the human insulin receptor. In D. melanogaster embryos and cell lines, the insulin receptor contains insulin-binding alpha subunits of 110 or 120 kilodaltons (kDa), a 95-kDa beta subunit that is phosphorylated on tyrosine in response to insulin in intact cells and in vitro, and a 170-kDa protein that may be an incompletely processed receptor. All of the components are synthesized from a proreceptor, joined by disulfide bonds, and exposed on the cell surface. The beta subunit is recognized by an antipeptide antibody elicited to amino acids 1142 to 1162 of the human insulin proreceptor, and the alpha subunit is recognized by an antipeptide antibody elicited to amino acids 702 to 723 of the human proreceptor. Of the polypeptide ligands tested, only insulin reacts with the D. melanogaster receptor. Insulinlike growth factors type I and II, epidermal growth factor, and the silkworm insulinlike prothoracicotropic hormone are unable to stimulate autophosphorylation. Thus despite the evolutionary divergence of vertebrates and invertebrates, the essential features of the structure and intrinsic functions of the insulin receptor have been remarkably conserved (Fernandez-Almonacid, 1987).

The cloning and primary structure of the Drosophila Insulin receptor gene (InR), functional expression of the predicted polypeptide, and the isolation of mutations in the InR locus are reported. These data indicate that the structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The InR proreceptor [M(r) 280 kDa] is processed proteolytically to generate an insulin-binding alpha subunit [M(r) 120 kDa] and a beta subunit [M(r) 170 kDa] with protein tyrosine kinase domain. The InR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension that contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage which leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90 and a free 60 kDa C-terminus) from the InR gene. These subunits assemble into mature InR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation of both types of beta subunits, which in turn allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of InR have different signaling potentials. Finally, loss of function mutations in the InR gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, render pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of InR appears to be required in the embryonic epidermis and nervous system among others, since development of the cuticle, as well as the peripheral and central nervous systems are all affected by InR mutations (Fernandez, 1995).

The Insulin-like receptor (InR) gene is strikingly homologous to the human receptor, exhibiting the same alpha2beta2 subunit structure and containing a ligand-activated tyrosine kinase in its cytoplasmic domain. Chemical mutagenesis was used to induce mutations in the InR gene and identified six independent mutations that led to a loss of expression or function of the receptor protein. These mutations are recessive, embryonic, or early larval lethals, but some alleles exhibit heteroallelic complementation to yield adults with a severe developmental delay (10 days), growth-deficiency, female-sterile phenotype. Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase. The growth deficiency appears to be due to a reduction in cell number, suggesting a role for Inr in regulation of cell proliferation during development. The phenotype is reminiscent of those seen in syndromes of insulin-resistance or IGF-I and IGF-I receptor deficiencies in higher organisms, suggesting a conserved function for this growth factor family in the regulation of growth and body size (Chen, 1996).

TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth

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/gigas 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;inr or 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).

To distinguish between these two possibilities, cells were generated that were doubly mutant for null alleles of PTEN and TSC1. PTEN is a negative regulator of the InR-PI3K-Akt pathway, and loss of PTEN results in increased Akt activity and cellular growth. It was reasoned that if TSC1 acts downstream from Akt within the InR-PI3K-Akt pathway, it might be expected that PTEN;TSC1 double-mutant cells would show a similar cell-size phenotype to either single mutant. However, if TSC1 acts parallel to the InR-PI3K-Akt the pathway, it might be expected that PTEN;TSC1 double-mutant cells would show additive effects on cell size as compared with each single mutant. PTEN;TSC1 double-mutant photoreceptors are 2.9 times the size of wild-type cells, as compared with 1.9 for PTEN - and 1.8 for TSC1 -. This result strongly suggests that the TSC genes function in a parallel pathway that converges on the insulin pathway at a point downstream from Akt (Gao, 2001).

In the course of these studies, a striking genetic interaction between the TSC genes and inr mutations was observed. Flies homozygous for a strong loss-of-function inr allele, are larval lethal. However, homozygous inr mutant flies that are heterozygous for TSC1 can survive to adults, suggesting that a mere 50% reduction in the dosage of the TSC1 gene can rescue the developmental arrest of an inr mutant (Gao, 2001).

Similarly, heterozygosity of TSC2 is sufficient to rescue the lethality of another inr mutant. Flies carrying the allelic combination inr353/inrl(3)05545 are 100% lethal. However, approximately 39% of inr353/inrl(3)05545 flies that are heterozygous for TSC2 can survive to adults. Taken together, these results provide convincing in vivo evidence that the TSC genes are negative regulators of insulin signaling in development (Gao, 2001).

A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function

The Drosophila gene insulin-like receptor (InR) is homologous to mammalian insulin receptors as well as to C. elegans daf-2, a signal transducer regulating worm dauer formation and adult longevity. A heteroallelic, hypomorphic genotype of mutant InR is described that yields dwarf females with up to an 85% extension of adult longevity and dwarf males with reduced late age-specific mortality. Treatment of the long-lived InR dwarfs with a juvenile hormone analog restores life expectancy toward that of wild-type controls. It is concluded that juvenile hormone deficiency, which results from InR signal pathway mutation, is sufficient to extend life-span, and that in flies, insulin-like ligands nonautonomously mediate aging through retardation of growth or activation of specific endocrine tissue (Tatar, 2001).

Molecular similarity between fly InR and worm daf-2 suggests that mutants of InR in flies should affect adult life-span, as do mutants of daf-2 in worms. InR and daf-2 are members of the insulin receptor family with homology to mammalian insulin and insulin-like growth factor-1 (IGF-1) receptors. Worms carrying temperature-sensitive mutations in daf-2 form dauers at high temperatures, but at lower temperatures develop directly into adults with extended longevity and resistance to exogenous stress. Genotypes homozygous for mutant InR have been reported to be lethal, but several heteroallelic combinations of InR alleles produce viable, dwarf adults that are slow to develop: InREC34/InRE19 and InRGC25/InRE19, and InRE19/InRp5545. In addition, InRE19/InRE19 is viable and dwarf, once crossed into a new isogenic background. Dwarf females eclose with extremely immature ovaries, and the egg chambers of young adults remain previtellogenic (Tatar, 2001).

Measurement of INR kinase activity indicates that the InRp5545 and InRE19 alleles both confer loss of INR function. Basal activity of heterozygotes +/InRE19 and +/InRp5545 was 45% of that of the wild type. Insulin stimulation increases kinase activity of INR from +/+ and +/InRE19 flies by 60%, but only by 26% from +/InRp5545 flies. Basal kinase activity of INR from InRE19/InRE19 and from InRp5545/InRE19 flies is 14% and 11% of that of the wild type, respectively; neither type is stimulated by insulin. InRp5545 is a P-element insertion in exon-1; the molecular lesion of InRE19 has yet to be identified, but it does not appear to occur in the known coding region of the gene (Tatar, 2001).

Life tables of InR mutant adults were compared to concurrent cohorts of a wild-type coisogenic strain. Dwarfs of InREC34/InRE19 and InRGC25/InRE19 are short-lived. Dwarf InRE19/InRE19 and nondwarf +/InRp5545 have moderately reduced survival; nondwarf +/InRE19 individuals are normal. In contrast, females of InRp5545/InRE19 are 85% longer lived than wild-type controls and overall present reduced age-specific mortality. The life-span of female D. melanogaster is also extended by mutation of the insulin receptor substrate homolog chico. Survivorship among male InR genotypes follows the pattern observed for females. Relative to the wild type, InRp5545/InRE19 males exhibit high mortality as early adults, but because of reduced mortality at late ages, dwarf life expectancy at 10 days is 43% greater than that of controls. It is likely that not all InR alleles increase longevity because the gene is highly pleiotropic, with some alleles producing developmental defects that carry over to the adult stage, which counterbalance positive effects of the allele upon aging (Tatar, 2001).

The fact that InR mutants are nonvitellogenic suggests a plausible mechanism for the extended longevity of InRp5545/InRE19 flies. Drosophila overwinter as adults in a reproductive diapause where egg development is arrested at previtellogenic stages. In many insects, including Drosophila, reproductive diapause is proximally controlled through down-regulation of juvenile hormone (JH) synthesis by the corpora allata (CA). Ovaries of InR dwarf females morphologically resemble ovaries of diapause wild-type flies, and exogenous application of the JH analog methoprene to dwarf females initiates vitellogenesis. Females of InRE19/InRE19 respond to a single treatment of methoprene in a dose-dependent manner, but females of InRp5545/InRE19 require continuous exposure to hormone to induce any vitellogenesis. Direct assay of adult JH synthesis verified that CA activity is reduced in InR dwarfs to about 23% of the wild-type level. Because reduced JH synthesis is seen in InRE19/InRE19 flies, which exhibit normal life-span, as well as in long-lived InRp5545/InRE19 flies, the simple lack of JH may not be enough to extend longevity (Tatar, 2001).

Loss of corpora allata JH accounts for dwarf infertility. Mutation of InR may increase longevity because infertility reduces allocation of metabolic resources to reproduction and frees resources for somatic maintenance or because reduced JH in mutant flies induces specific physiological mechanisms of somatic persistence normally expressed during adult reproductive diapause. Adult D. melanogaster in reproductive diapause age at negligible rates and are stress resistant; these traits are reversed by treatment with methoprene. Extended survival is characteristic of adult reproductive diapause in acridid grasshoppers and in the monarch butterfly, and surgical ablation of the corpora allata to eliminate adult JH synthesis induces both diapause and increased longevity. Consistent with the notion that reduced JH synthesis can directly extend life-span, InR dwarf flies show somatic physiological changes: (1) triglycerides are elevated fourfold (F = 32.2, P < 0.001), as observed in diapause D. triauraria and in dwarf D. melanogaster mutant for chico, and (2) Cu/Zn-superoxide dismutase concentration is increased twofold, as is characteristic of long-lived mutants of C. elegans. Measured in young adults, no difference in mass-specific metabolic rate is detected. It is suggested that infertility need not be the direct cause of slowed aging in InR mutants; JH may simply control both fertility and life-span (Tatar, 2001).

To test directly whether JH modulates survival in InRp5545/InRE19 female dwarfs, a test was made of whether treatment with methoprene restores wild-type longevity to these mutants, even if it does not fully restore fertility. In concurrent trials of dwarf and wild-type flies, survival of methoprene-treated InRp5545/InRE19 females is reduced toward the level observed in coisogenic controls. This rescue is physiological rather than toxicological because, in wild-type controls, methoprene produces no significant change relative to ethanol-treated flies (Tatar, 2001).

The InR pathway may alter endocrine function in two ways. Adult CA is derived from neurosecretory tissue of the larval ring gland. Adult dwarf CA may be immature upon metamorphosis as a result of cell autonomous effects of InR upon the development of neuroendocrine cells. A second way InR may alter endocrine function is that JH secretion by CA may be impaired by reduced neuropeptide transmission in the adult brain, due to a reduction of INR function in brain areas where it is normally expressed (Tatar, 2001).

In C. elegans, the insulin/IGF-1 pathway influences dauer formation, fertility, and aging in part through nonautonomous, secondary signaling; sterility is not required for extended longevity in C. elegans because some long-lived daf-2 are fully fertile. For Drosophila, InR affects neurosecretory tissue specialized for secretion of juvenile hormone. Therefore, mutations in the insulin signaling pathway in flies autonomously affect cell proliferation, growth, and body size, but nonautonomously affect diapause, reproduction, and life-span through effects upon specific neuroendocrine cells. Deficiency in a juvenoid-like hormone signal in worms and in flies may extend longevity because its absence leads to the inappropriate expression of parallel physiological programs normally reserved for dauer or diapause (Tatar, 2001).

This invertebrate model may have parallels with mammalian aging. Ames and Snell mice are mutant for the genes Prop-1 or Pit-1, respectively, and are defective for pituitary development. Consequently, they are deficient in growth hormone, prolactin, and thyroid-stimulating hormone, leading to hypothyroidism and presumably reduced synthesis of thyroxin, a retinoid hormone with potential functional similarity to JH. These mice are phenotypically dwarf, mildly obese, and long-lived. A remarkably similar phenotype is observed in mice lacking insulin receptor function in the central nervous system or those lacking the chico homolog, IRS-2, in all tissues: increased fat mass and infertility with accompanying neuroendocrine deficiency. Although effects on life-span in these mice remain to be determined, the concordance of phenotypes suggests that insulin signaling may be central to a common mechanism that exists across taxa for the neuroendocrine regulation of metabolism and the reproductive state, and their associated consequences upon aging (Tatar, 2001).

Extension of life-span by loss of Chico, a Drosophila Insulin receptor substrate protein

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).

PDK1 regulates growth through Akt and S6K in Drosophila

The insulin/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt/PKB. Although genetic analysis in C. elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have suggested that PDK1 has additional targets. In Drosophila, dPDK1 (FlyBase name: Protein kinase 61C) controls cellular and organism growth by activating Akt1 and S6 kinase, dS6K. Furthermore, dPDK1 genetically interacts with dRSK but not with dPKN (FlyBase name: Protein kinase related to protein kinase N), encoding two substrates of PDK1 in vitro. Thus, the results suggest that dPDK1 is required for dRSK but not dPKN activation and that it regulates insulin-mediated growth through two main effector branches, dAkt and dS6K (Rintelen, 2001).

The pronounced effect of loss of dPDK1 function on head size suggests that it is a dominant constituent in the dInr pathway. To test this possibility, the ability of complete and partial loss-of-function alleles of dPDK1 to reverse phenotypes caused by either overexpression of dInr or by mutations in dPTEN, the 3-phosphatidylinositide phosphatase, was evaluated. Overexpression of a wild-type dInr cDNA under the control of GMR-Gal4 leads to a marked increase in eye size and a slightly rough eye surface, an effect dominantly suppressed by removing one copy of dPDK1. Further reduction of dPDK1 function by the dPDK11/4 heteroallelic combination reduces the eye to almost wild-type size, suggesting that the amount of dPDK1 protein is rate-limiting for the dInr overgrowth phenotype. Null mutations in dPTEN cause lethality, and removal of dPTEN function in clones stimulates cell autonomous growth, suggesting that increased levels of PIP3 promote growth and are the likely cause of lethality. Thus, if dPDK1 is an essential target of PIP3, mutations in dPDK1 may suppress the dPTEN phenotype. Surprisingly, some dPTEN/dPDK1 double mutant flies survive to adulthood, indicating that the presumed PIP3-induced lethality is primarily caused by the hyperactivation of dPDK1 or of one of its targets (Rintelen, 2001).

These results show that dPDK1 is an essential component in the insulin signaling pathway in the control of cell growth and body size through its two substrates, dAkt and dS6K. These results are distinct from the genetic evidence in C. elegans where Akt is the primary target of PDK1 in dauer formation. Because mutations in the insulin signaling pathway do not show an autonomous alteration of cell size in C. elegans, the regulation of the rate of protein synthesis through S6K does not seem to be a primary target of this pathway. However, the fact that dPDK1 may yet have additional substrates is suggested by the genetic interaction with dRSK gain-of-function mutations and because viable dPDK1 males are almost completely sterile. Although mutations in components of the insulin signaling pathway such as dInr, chico, Dp110/PI(3)K, and dAkt cause female sterility, male sterility is not observed. Further genetic dissection of dPDK1 function is required to determine the role of dPDK1 in male fertility. These findings in Drosophila are consistent with the absence of insulin growth factor-1-induced activation of S6K, Akt, and RSK in mammalian PDK1-/- embryonic stem cells, and therefore provide evidence for the functional conservation of branch points in kinase networks during evolution (Rintelen, 2001).

The Drosophila insulin/IGF receptor controls growth and size by modulating PtdInsP3 levels

Insulin/IGF signaling during development controls growth and size, possibly by coordinating the activities of the Ras and PI 3-kinase signaling pathways. In vertebrates, the IR and IGFR act through IRS1-IRS4 proteins, which are multifunctional adaptors that link insulin and IGF signaling to the Ras/MAPK and phosphoinositide 3'-kinase (PI 3-kinase) signaling pathways. The pleckstrin homology domain (PH) and phosphotyrosine binding domain (PTB) of the IRS proteins are believed to mediate binding to phosphoinositol phosphates and the juxtamembrane NPXY motif of IR/IGFR, respectively. Grb2 (Drosophila homolog Drk) is an adaptor protein containing SH2 and SH3 domains. It has been suggested that Grb2 may, via its binding to IRS, link insulin/IGF to the Ras/MAPK pathway and thereby control proliferation. The Drosophila homolog of the SH2 domain containing p85 PI 3-kinase adaptor subunit, p60, binds Chico/IRS and thereby recruits the p110 catalytic subunit of PI 3-kinase [which converts phosphoinositol(4,5)P2 (PtdIns(4,5)P2) into phosphoinositol(3,4,5)P3 (PtdIns(3,4,5)P3)] to the plasma membrane. The p110 PI 3-kinase belongs to the class I PI 3-kinases implicated in the metabolic effects of insulin. The classical effectors that mediate the biological outcomes of insulin and IGF downstream of IRS have been divided into two functional branches: the Ras/MAPK proliferation pathway, and the PI 3-kinase metabolic, growth and survival pathway (Oldham, 2002).

To analyze the role of the different domains of Chico/IRS under physiological conditions, a panel of effector site mutants was created in a genomic rescue construct for chico that disrupts the PH or the PTB domains or the putative binding sites of Drk/Grb2 and p60. The constructs include the cis-regulatory sequences that permit expression of chico in its normal spatial and temporal pattern. The wild-type chico construct fully restores the defects of chico homozygous null mutants. In this manner, the effector site mutants were assayed for the ability to rescue the three different phenotypes associated with complete loss of Chico function: body size reduction, female sterility and lipid alterations. The Drk/Grb2 consensus binding site mutant is able to fully rescue the reduced weight to the same extent as the wild-type rescue construct. Therefore, the presence of a functional Drk binding site in Chico and thus the link to the activation of the Ras/MAPK kinase pathway is not required for its wild-type function. In contrast, the PH and PTB domain mutants and the double p60 PI 3-kinase binding site mutant were unable to rescue the reduced body weight. The latter result is surprising because InR contains additional functional PI 3-kinase binding sites in its C-terminal tail, an extension shared only with the C. elegans InR homolog, Daf-2, and not the mammalian IR or IGFR. This suggests that the presence of additional p60 binding sites in the InR C-terminal tail is not sufficient in vivo to mediate wild-type levels of growth and proliferation in the absence of the Chico p60 PI 3-kinase binding sites and that the InR C-terminal tail may contribute only low levels of PI 3-kinase signaling. Although the PTB domain mutant fails to restore normal body weight, it rescues the female sterility associated with the loss of Chico function. With the exception of the full rescue of the lipid accumulation observed in Drk/Grb2 mutant, all the other effectors only partially restore the change in lipid accumulation (Oldham, 2002).

To test whether increasing PtdInsP3 levels in an InR or PI 3-kinase p110 mutant background is sufficient to restore growth, the function of a negative regulator of the insulin pathway was eliminated. The 3'-phosphoinositol-specific lipid phosphatase, PTEN acts as a negative regulator of the PI 3-kinase pathway by converting PtdInsP3 generated by PI 3-kinase into PtdInsP2. Used were a null (Pten2L117) and a hypomorphic (Pten2L100) allele of Pten, identified in a screen for genes involved in growth control. As shown by HPLC analysis of the phospholipids in extracts of Pten mutant larvae, the loss of PTEN function results in a 2-fold increase in PtdInsP3 levels. This is consistent with the increase in PtdInsP3 seen in Pten-deleted murine fibroblasts. One prominent biological effect of these increased PtdInsP3 levels in Drosophila is a substantial increase in size in both larvae and pupae. To test whether loss of PTEN function, and consequently increased PtdInsP3 levels, is sufficient to restore growth or viability in InR null mutants, InR and Pten double mutants were generated by creating mosaic animals using the eyeless-Flipase (eyFlp) tissue-specific recombination system. In such animals, the head consists of homozygous mutant tissue, whereas the rest of the body is heterozygous for the same mutation. While loss of PTEN function (Pten2L117) in the head results in a fly with a disproportionately larger head (with more and larger cells), loss of InR function (InR327) results in flies with smaller heads (pinhead) compared to the wild type. Heads doubly mutant for Pten2L117and InR327, however, are almost the size of heads singly mutant for Pten2L117. Also, two different lethal heteroallelic InR combinations (InR304/InR327 or InR304/InR25), which arrest at the second larval instar stage, develop to the pupal stage (15%-17% of 33% expected) and even to pharate adults in the presence of reduced PTEN levels (Pten2L117/Pten2L100). These results demonstrate that complete loss of PTEN function can largely substitute for InR-mediated growth and proliferation in the absence of InR function and that the Ras/MAPK pathway plays little or no role in the InR mediated control of cell growth. This notion is further supported by the observation that complete loss of InR function in the compound eye does not result in a loss of photoreceptors, a hallmark of loss of Ras pathway function (Oldham, 2002).

The rescue of lethal, null InR mutant combinations to near viability by reducing PTEN activity strengthens the argument that a PtdInsP3-dependent signaling pathway is the primary effector for InR-derived growth and proliferation. In support of this observation, PI 3-kinase and Akt have been isolated as retroviral oncogenes, suggesting that activation of PI 3-kinase and Akt is sufficient to mediate growth, proliferation, and oncogenesis in vertebrate systems. In Drosophila and mammals, overexpression of PI 3-kinase causes increased growth; but this is not sufficient for proliferation as is the removal of Pten. From this premise, it has been proposed that PI 3-kinase and PTEN regulate similar yet distinct pathways. Alternatively, it is possible that they do function uniquely in the same pathway and that the difference may be due to altered location and function because of overexpression, or to differential feedback of PI 3-kinase versus PTEN. For example, since PI 3-kinase has been shown to act as a serine/threonine protein kinase on IRS, this may have a negative feedback effect on the insulin pathway that might not be evident in Pten loss-of-function mutations. Nevertheless, PI 3-kinase is absolutely critical in controlling size because using an allelic series of PI 3-kinase mutants in combination with the ey-Flp sytem resulted in a range of different head sizes. Furthermore, expressing an activated and dominant-negative form of PI 3-kinase in Drosophila imaginal discs or the heart of the mouse also leads to a corresponding increase or decrease in cell and organ size. Thus, the PI 3-kinase/PTEN cycle can be considered a dedicated growth rheostat, and the InR pathway is an evolutionary conserved module for regulating the range of growth and size (Oldham, 2002).

Loss of PTEN function results in a metabolically similar phenotype as loss of murine PTP1B (Ptpn1), an IR-specific tyrosine phosphatase, in that hyperactivation of the IR pathway causes resistance to high-fat-diet-induced obesity because of increased basal metabolism. These metabolic lipid effects have likely been conserved during evolution because the increased lipid levels in chico mutants are reminiscent of the enhanced lipid content in Irs2 deleted and NIRKO mice (Oldham, 2002).

Collectively, these data firmly establish Drosophila as a valid model organism for the study of metabolic diseases like diabetes and obesity as well as for the study of growth disorders like cancer. Pten mutant flies are larger in size due to increased cell size and number, but have a corresponding decrease in energy stores, a situation completely opposite that of mutations in positive components of the insulin signaling pathway like InR, chico, PI 3-kinase, and dAkt. These large viable Pten mutants show that a reduction of PTEN function is sufficient for increased organism size. This fact suggests that the four-fold size difference between viable InR and Pten mutants can simply be controlled by the amount of PtdInsP3 and this phenomenon may possibly be extended to vertebrate size regulation. Thus, in Drosophila, the InR/PI 3-kinase/PTEN pathway combines both metabolism and growth control into one pathway that later diverged into two separate, yet interacting systems in mammals (Oldham, 2002).

Insulin regulation of heart function in aging fruit flies

Insulin-IGF receptor (InR) signaling has a conserved role in regulating lifespan, but little is known about the genetic control of declining organ function. This study describes progressive changes of heart function in aging fruit flies: from one to seven weeks of a fly's age, the resting heart rate decreases and the rate of stress-induced heart failure increases. These age-related changes are minimized or absent in long-lived flies when systemic levels of insulin-like peptides are reduced and by mutations of the only receptor, InR, or its substrate, Chico. Moreover, interfering with InR signaling exclusively in the heart, by overexpressing the phosphatase PTEN or the forkhead transcription factor FOXO, prevents the decline in cardiac performance with age. Thus, insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its systemic effect on lifespan. The aging fly heart is a model for studying the genetics of age-sensitive organ-specific pathology (Wessells, 2004).

Direct control of germline stem cell division and cyst growth by neural insulin in Drosophila

Stem cells reside in specialized niches that provide signals required for their maintenance and division. Tissue-extrinsic signals can also modify stem cell activity, although this is poorly understood. This study reports that neural-derived Drosophila insulin-like peptides (DILPs) directly regulate germline stem cell division rate, demonstrating that signals mediating the ovarian response to nutritional input can modify stem cell activity in a niche-independent manner. A crucial direct role is demonstrated for DILPs in controlling germline cyst growth and vitellogenesis (LaFever, 2005).

Germline and somatic stem cells support oogenesis throughout adult life in Drosophila. Germline stem cells (GSCs) reside within a specialized niche where they are exposed to a unique combination of signals required for stem cell function. However, GSCs are also controlled by tissue-extrinsic signals, such as Drosophila insulin-like peptides (DILPs), which mediate the ovarian response to nutrition. On a protein-rich diet, germline and somatic stem cells have high division rates, and their progeny exhibit high division and development rates. On a protein-poor diet or under reduced insulin signaling, rates of division and development are reduced, and progression through vitellogenesis is blocked. It remains unclear, however, how DILPs control the response of GSCs in coordination with their differentiating progeny and with follicle cells (LaFever, 2005).

In adult females, DILPs are produced in two clusters of medial neurosecretory cells in the brain, and stage 10 follicle cells express dilp5 mRNA. Ablation of brain DILP-producing cells results in reduced egg production rates and a partial block in vitellogenesis. To examine the role of the brain DILP-producing cells in previtellogenic stages, they were ablated and follicle cell proliferation rates were measured. Females missing brain DILP-producing cells (ablated) have a severely impaired ability to up-regulate follicle cell proliferation in response to a protein-rich diet. The rate of germline development is reduced in coordination with follicle cell divisions because no abnormalities are observed in previtellogenic egg chambers. Ablation of DILP-producing cells reduces the size of eclosed adults and delays development. Ablated females in which these developmental defects are rescued by an hs-dilp2 transgene expressed during larval stages show a reduced follicle cell proliferation rate, comparable to that of nonrescued, ablated females. Thus, the impaired response to a protein-rich diet is not a secondary consequence of the developmental defects. Moreover, the 2.3-fold delay caused by ablation of brain DILP-producing cells is very similar to that caused by blocking reception of DILP signals by the germ line. This indicates that the brain is the major source of DILPs that determine the rate of egg chamber development with little, if any, contribution from dilp5-expressing follicle cells (LaFever, 2005).

To examine whether DILPs control the rate of germline development directly or indirectly, germline cysts unable to respond to DILPs were created by inducing Drosophila insulin receptor (dinr) mutant clones using the flipase (FLP)/FLP-recognition target (FRT) technique. Germline cysts homozygous for dinr339, a genetic null allele, had normal morphology and correct cell number; however, 83% of these cysts were developmentally delayed, showing markedly decreased size relative to neighboring wild-type egg chambers. Further quantification of these data showed a 2.4-fold delay in the development of dinr339 cysts. Similar results were obtained for germline cysts homozygous for dinrE19 and dinr353, which are viable hypomorphic alleles; 78% and 64% of dinrE19 and dinr353 cysts, respectively, were developmentally delayed. These results reveal that dinr function is required cell autonomously for a normal rate of germline cyst development. Thus, the rate of cyst development is regulated by a DILP signal that is received directly (LaFever, 2005).

Progression through vitellogenesis requires DILP signaling; however, it has been unclear whether this role is direct. Reduced juvenile hormone levels are present in homozygous viable dinr mutants, and the block in yolk uptake in these mutants can be partially bypassed by treatment with methoprene, a juvenile hormone analog, suggesting an indirect role for DILPs in promoting vitellogenesis. To specifically address whether direct activation of germline cysts by DILPs is required for vitellogenesis, mosaic ovarioles were examined in which the entire germ line was homozygous dinr mutant for the ability of their egg chambers to undergo vitellogenesis. All egg chambers containing dinr339 or dinrE19 homozygous mutant cysts failed to progress through vitellogenesis and degenerated. In the case of dinr353, the allele with the higher level of dinr activity, only one out of six egg chambers containing homozygous mutant cysts failed to undergo vitellogenesis. These results suggest that the level of insulin signaling within the germ line controls vitellogenesis, revealing a direct role for DILPs in this process. Moreover, complete loss of dinr function in the germ line results in a complete block in vitellogenesis, whereas this block is partial upon ablation of brain DILP-producing cells. Thus, DILP5 expressed in stage 10 follicle cells likely signals in combination with brain DILPs to regulate vitellogenesis (LaFever, 2005).

It was next asked whether DILPs control GSC division rate directly by binding to receptors on their surface (a cell-autonomous requirement for dinr in GSCs) or indirectly by regulating signals produced by niche cells (a non-cell-autonomous requirement). dinr mosaic ovarioles containing one wild-type and one mutant GSC were examined and the number of wild-type versus mutant cystoblasts and cysts present in their germaria was counted. Because each cystoblast or cyst corresponds to one GSC division, the ratio of mutant to wild-type cystoblasts and cysts is a measure of their relative division rates. For dinr339 homozygous mutant GSCs, a relative division rate of 0.31 was found, whereas, for wild-type GSCs, it was 0.90. Similarly, the relative division rates of dinr353 and dinrE19 GSCs were 0.55 and 0.65, respectively. Thus, dinr homozygous mutant GSCs divide more slowly than wild-type GSCs, and GSC division rate appears sensitive to the level of dinr activity. These results demonstrate that GSCs directly receive the DILP signal to regulate their division rate without mediation by the stem cell niche (LaFever, 2005).

Germline and somatic cells respond to nutritional status in a coordinated manner; however, it is unclear whether somatic cells receive the DILP signal directly (a cell-autonomous role of dinr in follicle cell proliferation) as does the germ line, or indirectly through secondary signals (a non-cell-autonomous role). The percentages of dinr mutant and control follicle cells were measured in mosaic ovarioles carrying one wild-type and one dinr mutant somatic stem cell. If follicle cells receive the DILP signal directly, the reduced level of insulin signaling in dinr mutant follicle cells should result in lower rates of proliferation (i.e., fewer mutant than control follicle cells should be observed), whereas if they receive the signal indirectly, the proliferation rates should be similar. In dinrE19 mosaic ovarioles, 51% of follicle cells were wild-type and 49% were mutant, indicating similar proliferation rates. dinr mutant follicle cells appeared to enter the endoreplicative cycle normally, but pycnotic (degenerating) nuclei and cell death were observed within dinrE19 and dinr339 mutant follicle cell clones starting at stage 8. These results reveal that although a reduction in dinr activity delays germline cyst development cell autonomously, it does not cause a cell-autonomous reduction in follicle cell proliferation rate. Furthermore, in ovarioles carrying a fully dinr mutant germ line, excess follicle cells were not observed, showing that proliferation of surrounding wild-type follicle cells remains coordinated with germline growth. These results suggest that follicle cells respond indirectly to increased DILP levels through a secondary signal from the germ line. Similar degrees of coordination between germ line and soma have been observed in the presence of developmentally delayed dMyc mutant germline clones (LaFever, 2005).

These data demonstrate that tissue-extrinsic DILP signals can directly modify GSC proliferative activity, acting in parallel to signals from their niche. Evidence is provided that, in addition to its previously reported indirect roles in Drosophila and mammals through secondary hormonal signals, insulin signaling plays a crucial direct role during Drosophila oogenesis in regulating not only GSC division rate but also germline cyst development rate and progression through vitellogenesis. Insulin may, therefore, have important direct roles in mammalian oogenesis. Finally, the data suggest that the coordinated response of germline and somatic cells to nutrition involves communication between these tissues. These results have broad significance, in light of the long-known effects of nutrition on human fertility and of the high degree of conservation of insulin signaling functions (LaFever, 2005).

Insulin levels control female germline stem cell maintenance via the niche in Drosophila

Stem cell maintenance depends on local signals provided by specialized microenvironments, or niches, in which they reside. The potential role of systemic factors in stem cell maintenance, however, has remained largely unexplored. This study shows that insulin signaling integrates the effects of diet and age on germline stem cell (GSC) maintenance through the dual regulation of cap cell number (via Notch signaling) and cap cell-GSC interaction (via E-cadherin) and that the normal process of GSC and niche cell loss that occurs with age can be suppressed by increased levels of insulin-like peptides. These results underscore the importance of systemic factors for the regulation of stem cell niches and, thereby, of stem cell numbers (Hsu, 2009).

The stem cell microenvironment (niche) controls stem cells, and niche aging leads to stem cell decline. The Drosophila germline stem cell (GSC) niche includes terminal filament cells, cap cells, and escort stem cells, and GSC fate and activity require direct contact with cap cells and exposure to niche-derived signals. GSCs also respond to systemic signals, such as Drosophila insulin-like peptides (DILPs), which directly modulate their proliferation. Increased age leads to decreased niche size and signaling and GSC loss. The molecular basis for age-dependent changes in the niche, however, remains poorly understood (Hsu, 2009).

Because diet influences aging, its effects on GSC maintenance were examined, exploiting the fact that GSCs can be unambiguously identified by their anteriorly anchored fusome (a membranous cytoskeletal structure) and by their juxtaposition to cap cells. A decrease was observed in GSC numbers in well-fed females over time. In females on a poor diet, however, the rate of GSC loss was significantly increased (Hsu, 2009).

Insulin secretion and signaling respond to diet and diminish in aging humans. Using a phosphoinositide 3-kinase reporter, reduced insulin signaling was found in older ovaries. To address if GSC maintenance requires insulin signaling, GSC numbers were measured in Drosophila insulin receptor (dinr) mutants. The dinr339/dinrE19 females contain slightly fewer GSCs at eclosion and lose them significantly faster than controls. GSC death was not observed in dinr339/dinrE19 or control germaria, suggesting that GSC loss results from differentiation (Hsu, 2009).

The chico1 homozygotes, which lack insulin receptor substrate, a major insulin pathway component, also show increased GSC loss. Thus, insulin signaling controls GSC maintenance. Next, whether DILP expression in germarial somatic cells could counteract the wild-type age-dependent GSC loss was tested. The c587-GAL4 driver was used to express a UAS-dilp2 transgene, encoding the DILP most closely related to human insulin, and thereby increase the local levels of insulin-like signals. GSC loss on rich and poor diets was significantly suppressed by DILP2 overexpression, although this was less pronounced in 4-week-old females on a poor diet. The less effective rescue on a poor diet could potentially be attributable to lower expression of the c587-GAL4 driver, to the actions of additional diet-dependent signals, or to a combination thereof. Nevertheless, these results suggest that the normal GSC loss observed in wild-type females as their age increases results largely from reduced insulin signaling (Hsu, 2009).

DILPs control GSC division directly, leading to a cell-autonomous dinr requirement. It was therefore asked whether dinr is required within GSCs for their maintenance. In genetic mosaics, homozygous dinr339 or dinrE19 GSCs are not lost at a higher rate than control GSCs, demonstrating that DILPs do not promote GSC maintenance directly (Hsu, 2009).

It was next hypothesized that insulin signaling may regulate GSC fate via the niche. Indeed, expression of wild-type dinr in somatic cells of dinr339/dinrE19 germaria rescued GSC loss. To examine dinr339/dinrE19 niche structure, terminal filament and cap cells were counted. Terminal filament cell numbers in dinr339/dinrE19 and control females are similar. In contrast, dinr339/dinrE19 females eclose with fewer cap cells and also lose them faster over time, suggesting that insulin signaling controls cap cell number during development and adulthood. Moreover, DILP2 overexpression suppresses the wild-type age-dependent cap cell number decrease. It is concluded that DILPs control GSC niche size and that the reduced cap cell numbers observed with increased female age at least in part reflect low insulin signaling levels (Hsu, 2009).

It was next asked whether DILPs control cap cell number directly. In mosaic germaria containing β-gal-negative dinr339 or control cap cells, the distribution (and average number) of β-gal-negative cap cells was indistinguishable, indicating that dinr does not control cap cell number cell autonomously. It is possible that a second cell type, such as terminal filament cells, produces an intermediate factor; alternatively, cap cells themselves may control their own maintenance via paracrine signaling (Hsu, 2009).

Notch signaling controls cap cell number during niche formation and in adults. Notch hyperactivation during development forms ectopic cap cells, leading to excess GSCs. Conversely, defective Notch signaling reduces niche size and GSC number. Notch activation is strongly detected in larval terminal filament and cap cells and is also detected in adult cap cells. Notch signaling was examined in dinr mutants using the E(spl)mβ-CD2 reporter. Every control germarium had strong CD2 expression in both terminal filament and cap cells. In contrast, CD2 levels were severely reduced in dinr339/dinrE19 germaria, indicating that insulin signaling controls Notch activation in the niche (Hsu, 2009).

It was next asked if the reduced cap cell number in dinr mutants was attributable to impaired Notch signaling. Weak hypomorphic dinrE19/dinr353 females have no reduction in GSC or cap cell number. Similarly, Notch heterozygotes (half the Notch dosage) have normal GSC and cap cell numbers. In contrast, dinrE19/dinr353 females heterozygous for Notch have significantly reduced GSC and cap cell numbers. A decrease in small cap cell number has been reported for Notch heterozygotes; this discrepancy may reflect slightly reduced insulin signaling in the latter study attributable to diet (Hsu, 2009).

To determine if Notch signaling is sufficient to rescue dinr defects, an activated form of Notch was expressed in the somatic cells of dinr339/dinrE19 germaria, and the GSC and cap cell loss phenotypes were rescued. These results and the genetic interaction between dinr and Notch are consistent with the insulin pathway acting upstream or in parallel to Notch. Nevertheless, the reduced Notch reporter levels in dinr mutants favor the model that insulin signaling leads to Notch activation, thereby controlling cap cell number and, indirectly, GSC maintenance (Hsu, 2009).

GSCs and terminal filament cells express the Delta ligand for Notch, and removal of Delta function from GSCs has been reported to affect niche activity. It was reasoned that dinr could be required in GSCs, terminal filament cells, the cap cell population, or a combination thereof to control Delta production and Notch activation. dinr mosaic germaria were examined in which all GSCs were dinr339 homozygous, and the number of cap cells in those germaria was indistinguishable from control numbers, suggesting that dinr is not required in GSCs for Notch signaling. DILPs may instead regulate Delta within terminal filament or cap cells or, alternatively, act via other intermediate signals to regulate Notch activation within the niche (Hsu, 2009).

Cap cell and GSC numbers correlate. Indeed, in germaria containing control β-gal-negative cap cells (control C1), total cap cell and GSC numbers are roughly proportional. Remarkably, despite similar cap cell numbers, a significant fraction of germaria in which dinr mutant cap cells are present contains fewer GSCs relative to control C1 or C2 (i.e., germaria without cap cell clones from dinr mosaics). Thus, although dinr does not control cap cell number per se autonomously, it is required within cap cells either for the optimal production and/or secretion of a GSC maintenance factor(s) or to promote GSC attachment (Hsu, 2009).

Niche-derived bone morphogenetic protein (BMP) signals directly stimulate GSCs to repress differentiation. To test if insulin signaling controls BMP pathway activation in GSCs, the Dad-lacZ reporter was used. Dad-lacZ levels in dinr339/dinrE19 and control females are indistinguishable, showing that dinr does not control BMP signaling. Insulin signaling in cap cells must therefore control another GSC maintenance signal and/or the cap cell-GSC association (Hsu, 2009).

To investigate if dinr controls the physical interaction between cap cells and GSCs, the percentage of dinr339 versus control cap cells directly contacting GSCs was measure in mosaic germaria. Indeed, 21% of dinr339 cap cells contact GSCs, compared with 50% of control cap cells, indicating that dinr339 cap cells have significantly reduced attachment to GSCs. These results suggest that insulin signaling in cap cells controls their association with GSCs. Alternatively, insulin signaling may regulate the production of a short-range GSC maintenance signal, such that only GSCs in contact with dinr mutant cap cells are affected (Hsu, 2009).

E-cadherin-mediated adhesion between cap cells and GSCs is required for retaining GSCs in the niche. Therefore E-cadherin levels were measured at the GSC-cap cell junction. In controls, it was found that E-cadherin levels vary with changes in the fusome, a membranous cytoskeletal structure. When the fusome is round, its predominant morphology, there is a higher intensity of E-cadherin at the junction, although when the fusome is elongated, the intensity is lower. The intensity of E-cadherin at the junction of cap cells with GSCs displaying elongated fusomes in dinr339/dinrE19 mutants is similar to that of control. In contrast, the round fusome GSC-cap cell junctions contain significantly lower E-cadherin levels in dinr mutants than in controls. These results suggest that insulin signaling influences E-cadherin levels at the GSC-cap cell junction and may explain the age-dependent E-cadherin reduction that contributes to GSC loss (Hsu, 2009).

These studies demonstrate that systemic insulin-like signals integrate inputs from diet and age to regulate GSC maintenance via the niche. Specifically, it is proposed that DILPs control cap cell number via Notch and also E-cadherin- mediated GSC retention within the niche. Because diet and insulin signaling control GSC proliferation, it is also likely that the proliferation decline reported in older females results from reduced insulin signaling. These results also provide insights into recent findings that systemic factors from young mice can restore Notch activation and skeletal muscle progenitor proliferation and regenerative capacity to old mice in heterochronic parabiotic pairings. Finally, the results are intriguing in light of the well-established connection between low insulin signaling, restricted diet, and extended lifespan and of studies in C. elegans suggesting that GSCs may have a negative effect on longevity. It is conceivable that excessive stem cell activity in general is deleterious and that slight decreases in stem cell number or activity with age as a result of reduced insulin signaling may actually promote longevity (Hsu, 2009).

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).

Insulin signalling regulates remating in female Drosophila

Mating rate is a major determinant of female lifespan and fitness, and is predicted to optimize at an intermediate level, beyond which superfluous matings are costly. In female Drosophila melanogaster, nutrition is a key regulator of mating rate but the underlying mechanism is unknown. The evolutionarily conserved insulin/insulin-like growth factor-like signalling (IIS) pathway is responsive to nutrition, and regulates development, metabolism, stress resistance, fecundity and lifespan. This study shows that inhibition of IIS, by ablation of Drosophila insulin-like peptide (DILP)-producing median neurosecretory cells, knockout of dilp2, dilp3 or dilp5 genes, expression of a dominant-negative DILP-receptor (InR) transgene or knockout of Lnk, results in reduced female remating rates. IIS-mediated regulation of female remating can occur independent of virgin receptivity, developmental defects, reduced body size or fecundity, and the receipt of the female receptivity-inhibiting male sex peptide. These results provide a likely mechanism by which females match remating rates to the perceived nutritional environment. The findings suggest that longevity-mediating genes could often have pleiotropic effects on remating rate. However, overexpression of the IIS-regulated transcription factor dFOXO in the fat body-which extends lifespan-does not affect remating rate. Thus, long life and reduced remating are not obligatorily coupled (Wigby, 2011).

The effects of IIS on female remating can - at least to some extent - act independently of SP, the major male-derived molecular effector of female receptivity. This finding is consistent with the lack of interaction effects between nutrition and SP on female mating rate found by Fricke (2010). These two major regulators of female remating, IIS and SP, are likely to signal the normal requirement for remating in response to factors that limit female reproduction, namely nutrients required to produce eggs and sperm required for fertilization. This dual mechanism for controlling remating, via IIS and SP, may enable female mating rate to most effectively match reproductive opportunities while avoiding costly superfluous matings (Wigby, 2011).

Females may benefit unconditionally from their first mating as they need to obtain sperm to fertilize eggs. Thus, the lack of effect of IIS on virgin receptivity may be because sexually mature females gain from a rapid first mating - and there is no benefit to delaying mating -- whatever may be the nutritional conditions. However, in D. melanogaster, as in many insects, a single mating fails to provide sufficient sperm to fertilize all the eggs produced over a lifetime, meaning that females must remate to replenish sperm stores. A tighter calibration of nutrition with remating rate may be beneficial following the first mating, because nutrition affects female fecundity and the rate of sperm use such that, under poor nutritional conditions, females will need to replenish stored sperm (i.e. mate) less frequently. Hence, the regulation of female remating receptivity in response to nutritional status is likely to be key for female fitness (Wigby, 2011).

The sexual behaviour of IIS mutant females broadly mimics that of females on a poor diet, which is consistent with the hypothesis that reduced IIS partly (though not wholly) mimics dietary restriction. Like reduced IIS, restriction of dietary nutrients can result in increased lifespan and decreased mating rates. Manipulating components of the IIS pathway, as performed in this study, could generate a mismatch between the perceived and real nutritional environment, resulting in potentially sub-optimal mating rates for a given rate of egg-laying. However, it is clear that there is no obligatory link between egg-laying and mating rate, because females that lack the ability to produce eggs display normal mating and remating behaviours. Moreover, this study shows that females can possess normal fecundity but show reduced mating rates under IIS suppression (Wigby, 2011)

Lifespan can be extended by genetic manipulations that reduce IIS, including several mutants used in this study (MNC-ablated; dilp2 and dilp2-3; InRDN; Lnk). However, lifespan can also be extended by reducing mating frequency. The results therefore highlight the importance of controlling mating rates in studies that investigate the genetics of ageing, to avoid confounding effects of differential sexual activity on lifespan. The discovery that several IIS manipulations that increase lifespan also increase the inter-mating interval raises an important potential confound regarding the conclusions of ageing studies in which flies are maintained in mixed sex groups. Reduced mating rates in experimental mutant lines could potentially confound ageing studies because females might live longer owing to reduced mating rates rather than as a direct effect of the genetic manipulations themselves. The solution to this potential confound is to control mating rates in lifespan studies in order to test for direct effects on lifespan. However, the results from the dFOXO experiment show that it is also possible to uncouple the regulation of female sexual behaviour and the regulation of lifespan, in accordance with the uncoupling of lifespan and fecundity. Thus, both behavioural and physiological aspects of reproduction can be uncoupled from lifespan extension under certain conditions (Wigby, 2011).

The effects of single dilp mutants on remating were, surprisingly, only marginally weaker than the effects of MNC ablation or dilp2-3 double mutants, despite the apparently weaker genetic intervention. However, ablation of the MNCs is incomplete, and DILP levels are reduced rather than abolished in the flies that were used. Moreover, there is compensation and synergism between DILPs such that knockouts of single dilp genes can affect the expression of one or more of the other dilps. For example, dilp2 and dilp2-3 mutant flies exhibit increased expression of dilp5, while dilp3 mutants exhibit reduced levels of dilp2 and dilp5 expression. Such effects could explain the relatively strong phenotypes of the single dilp knockouts in comparison with the dilp2-3 knockout and MNC-ablated females (Wigby, 2011).

The extracellular DILPs, the InR and the intracellular IIS component, Lnk, all regulate female remating rate, but it is currently unclear which downstream molecules are involved. A major downstream target of the IIS pathway is the transcription factor dFOXO, but no effect of fat body dFOXO expression was found on female mating. One possibility is that dFOXO mediates the effect of reduced IIS on remating rates in tissues other than the fat body. Another possibility is that the effect of IIS on remating rate occurs via the target of rapamycin (TOR) pathway. The TOR pathway senses amino acids and runs parallel to, and interacts with, IIS. The IIS and TOR pathways interact to control growth, and TOR signalling, like IIS, has been shown to regulate lifespan. Moreover, recent work shows that the TOR pathway is involved in mating-induced changes in diet choice, supporting the idea that TOR functions in the coordination of behavioural responses to mating and the nutritional environment. It will be important to investigate the mating behaviour of TOR-pathway mutants to determine whether this pathway is involved in the regulation of mating and whether the effects of IIS on female remating are mediated through TOR signalling. It will also be important to determine through which tissues IIS regulates remating (Wigby, 2011).

This work shows that components of the IIS pathway modulate sexual behaviour by significantly altering the receptivity of mated female D. melanogaster. Thus, a likely molecular basis is provided for the link between nutrition and sexual behaviour in insects, which is an important step in understanding the mechanisms underlying life-history traits and trade-offs. Reproduction and nutrition are linked across a broad range of taxa, including mammals, and many of the effects of IIS (e.g. on lifespan and fecundity) are highly evolutionarily conserved. It is concluded that the regulation of mating behaviour via IIS could be common among animals (Wigby, 2011).

Insulin signaling regulates neurite growth during metamorphic neuronal remodeling

Although the growth capacity of mature neurons is often limited, some neurons can shift through largely unknown mechanisms from stable maintenance growth to dynamic, organizational growth (e.g. to repair injury, or during development transitions). During insect metamorphosis, many terminally differentiated larval neurons undergo extensive remodeling, involving elimination of larval neurites and outgrowth and elaboration of adult-specific projections. This study shows in the fruit fly that a metamorphosis-specific increase in insulin signaling promotes neuronal growth and axon branching after prolonged stability during the larval stages. FOXO, a negative effector in the insulin signaling pathway, blocks metamorphic growth of peptidergic neurons that secrete the neuropeptides CCAP and bursicon. RNA interference and CCAP/bursicon cell-targeted expression of dominant-negative constructs for other components of the insulin signaling pathway (InR, Pi3K92E, Akt1, S6K) also partially suppresses the growth of the CCAP/bursicon neuron somata and neurite arbor. In contrast, expression of wild-type or constitutively active forms of InR, Pi3K92E, Akt1, Rheb, and TOR, as well as RNA interference for negative regulators of insulin signaling (PTEN, FOXO), stimulate overgrowth. Interestingly, InR displays little effect on larval CCAP/bursicon neuron growth, in contrast to its strong effects during metamorphosis. Manipulations of insulin signaling in many other peptidergic neurons revealed generalized growth stimulation during metamorphosis, but not during larval development. These findings reveal a fundamental shift in growth control mechanisms when mature, differentiated neurons enter a new phase of organizational growth. Moreover, they highlight strong evolutionarily conservation of insulin signaling in neuronal growth regulation (Gu, 2014).

It is well established that insulin/insulin-like signaling (IIS) is crucial for regulating cell growth and division in response to nutritional conditions in Drosophila. However, most studies have focused on growth of the body or individual organs, and comparatively little is known about the roles of IIS during neuronal development, particularly in later developmental stages. Drosophila InR transcripts are ubiquitously expressed throughout embryogenesis, but are concentrated in the nervous system after mid-embryogenesis and remain at high levels there through the adult stage. This suggests that IIS plays important roles in the post-embryonic nervous system. Recently, analysis of Drosophila motorneurons, mushroom body neurons, and IPCs has revealed important roles of PI3K and Rheb in synapse growth or axon branching. These studies revealed some growth regulatory functions of IIS in the CNS, but they have not explored whether the control of neuronal growth by IIS is temporally regulated (Gu, 2014).

This study has shown that IIS strongly stimulates organizational growth of neurons during metamorphosis, whereas the effects of IIS on larval neurons are comparatively modest. Recently, similar results have been reported in mushroom body neurons, in which the TOR pathway strongly promoted axon outgrowth of γ-neurons after metamorphic pruning. Expression of FOXO or reduction of InR function had no significant effect on larval growth of the CCAP/bursicon neurons, or on the soma size of many other larval neurons. Thus, while IIS has been shown to regulate motorneuron synapse expansion in larvae, the current findings indicate that IIS may not play a major role in regulating structural growth in many larval neurons. This is consistent with a recent report that concluded that the Drosophila larval CNS is insensitive to changes in IIS (Gu, 2014).

When InRact was used to activate IIS without ligand, a modest but significant increase was seen in the soma size of the more anterior CCAP/bursicon neurons during larval development. This result indicates that the IIS pathway is present and functional in these larval neurons, but the ligand for InR is either absent or inactive. During metamorphosis, unlike in larvae, downregulation of IIS by altering the level of InR or downstream components of the pathway significantly reduced CCAP/bursicon neuron growth. Thus, the results suggest that IIS is strongly upregulated during metamorphosis to support post-embryonic, organizational growth of diverse peptidergic neurons, and this activation may at least in part be due to the presence of as yet unidentified InR ligands during metamorphosis (Gu, 2014).

Attempts were to identify this proposed InR ligand source by eliminating, in turn, most of the known sources of systemic DILPs. None of these manipulations had any effect on metamorphic growth of the CCAP/bursicon neurons. These results are consistent with three possible mechanisms. First, there may be a compensatory IIS response to loss of some dilp genes. For example, a compensatory increase in fat body DILP expression has been observed in response to ablation of brain dilp genes. Second, the growth may be regulated by another systemic hormone (e.g. DILP8) that was not tested, or by residual DILP peptides in the RNAi knockdown animals. Third, a local insulin source may be responsible for stimulating metamorphic outgrowth of the CCAP/bursicon neurons. Consistent with this view, a recent report showed that DILPs secreted from glial cells were sufficient to reactivate neuroblasts during nutrient restriction without affecting body growth, while overexpression of seven dilp genes (dilp1-7) in the IPCs had no effect on neuroblast reactivation under the same conditions. It seems likely that glia or other local DILP sources play an important role in regulating metamorphic neuron growth, but further experiments will be needed to test this model (Gu, 2014).

When IIS was manipulated in the CCAP/bursicon neurons, changes were observed in cell body size (and sometimes shape) and in the extent of branching in the peripheral axon arbor. Although this study focused analysis of neurite growth on the peripheral axons, which are easily resolved in fillet preparations, corresponding changes were also observed in the size and complexity of the central CCAP/bursicon neuron arbor. These IIS manipulations (both upregulation or downregulation) resulted in the above structural changes as well as wing expansion defects, suggesting that the normal connectivity of the CCAP/bursicon neurons was required for proper functioning of this cellular network. This model is consistent with the observation of two subsets of morphologically distinct bursicon-expressing neurons (the BSEG and BAG neurons), which are activated sequentially to control central and peripheral aspects of wing expansion. The BSEG neurons project widely within the CNS to trigger wing expansion behavior as well as secretion of bursicon by the BAG neurons. In turn, the BAG neurons send axons into the periphery to secrete bursicon into the hemolymph to control the process of tanning in the external cuticle. Therefore, manipulation of IIS within these neurons, and the changes in morphology that result, may disrupt the wiring and function of this network. However, because the possibility cannot be ruled out that these IIS manipulations also altered neuronal excitability, synaptic transmission, or neuropeptide secretion, this study relied on measurements of cellular properties (and not wing expansion rates) when assessing the relative effects of different IIS manipulations on cell growth (Gu, 2014).

The results indicate that IIS is critical for organizational growth, which occurs during insect metamorphosis but is also seen during neuronal regeneration in other systems. However, the regenerative ability of many neurons is age-dependent and context-dependent; immature neurons possess a more robust regenerative capacity, while the regenerative potential of many mature neurons is largely reduced. In particular, the adult vertebrate CNS displays very limited regeneration, in marked contrast to the regeneration abilities displayed by the peripheral nervous system. Recent studies in mice suggest that age-dependent inactivation of mTOR contributes to the reduced regenerative capacity of adult corticospinal neurons, and activation of mTOR activity through PTEN deletion promoted robust growth of corticospinal tract axons in injured adult mice. The current genetic experiments demonstrate a requirement for activity of TOR, as well as several other IIS pathway components both upstream and downstream of TOR, in controlling organizational growth of many peptidergic neurons. This suggests that under certain conditions, the activation of IIS may be a crucial component of the conversion of mature neurons to a more embryonic-like state, in which reorganizational growth either after injury or as a function of developmental stage is possible. Given the strong evolutionary conservation of these systems and the powerful genetic tools available to identify novel regulatory interactions in Drosophila, studies on the control of organizational growth in this species hold great promise for revealing factors that are crucial for CNS regeneration (Gu, 2014).

A genetic strategy to measure circulating Drosophila insulin reveals genes regulating insulin production and secretion

Insulin is a major regulator of metabolism in metazoans, including the fruit fly Drosophila melanogaster. Genome-wide association studies (GWAS) suggest a genetic basis for reductions of both insulin sensitivity and insulin secretion, phenotypes commonly observed in humans with type 2 diabetes mellitus (T2DM). To identify molecular functions of genes linked to T2DM risk, a genetic tool was developed to measure insulin-like peptide 2 (Ilp2) levels in Drosophila, a model organism with superb experimental genetics. This system permitted sensitive quantification of circulating Ilp2, including measures of Ilp2 dynamics during fasting and re-feeding, and demonstration of adaptive Ilp2 secretion in response to insulin receptor haploinsufficiency. Tissue specific dissection of this reduced insulin signaling phenotype revealed a critical role for insulin signaling in specific peripheral tissues. Knockdown of the Drosophila orthologues of human T2DM risk genes, including GLIS3 and BCL11A, revealed roles of these Drosophila genes in Ilp2 production or secretion. Discovery of Drosophila mechanisms and regulators controlling in vivo insulin dynamics should accelerate functional dissection of diabetes genetics (Park, 2014. PubMed ID: 25101872).

Disruption of insulin signalling affects the neuroendocrine stress reaction in Drosophila females

Juvenile hormone (JH) and dopamine are involved in the stress response in insects. The insulin/insulin-like growth factor signalling pathway has also recently been found to be involved in the regulation of various processes, including stress tolerance. However, the relationships among the JH, dopamine and insulin signalling pathways remain unclear. The role of insulin signalling in the regulation of JH and dopamine metabolism under normal and heat stress conditions was investigated in Drosophila melanogaster females. Suppression of the insulin-like receptor (InR) in the corpus allatum, a specialised endocrine gland that synthesises JH, causes an increase in dopamine level and JH-hydrolysing activity and alters the activities of enzymes that produce as well as those that degrade dopamine [alkaline phosphatase (ALP), tyrosine hydroxylase (TH) and dopamine-dependent arylalkylamine N-acetyltransferase (DAT)]. It was also found that InR suppression in the corpus allatum modulates dopamine, ALP, TH and JH-hydrolysing activity in response to heat stress and that it decreases the fecundity of the flies. JH application restores dopamine metabolism and fecundity in females with decreased InR expression in the corpus allatum. These data provide evidence that the insulin/insulin-like growth factor signalling pathway regulates dopamine metabolism in females of D. melanogaster via the system of JH metabolism and that it affects the development of the neuroendocrine stress reaction and interacts with JH in the control of reproduction in this species (Rauschenbach, 2014).

A highly pleiotropic amino acid polymorphism in the Drosophila insulin receptor contributes to life history adaptation

Finding the specific nucleotides that underlie adaptive variation is a major goal in evolutionary biology, but polygenic traits pose a challenge because the complex genotype-phenotype relationship can obscure the effects of individual alleles. However, natural selection working in large wild populations can shift allele frequencies and indicate functional regions of the genome. Previous studies have shown that the two most common alleles of a complex amino acid insertion-deletion polymorphism in the Drosophila Insulin receptor show independent, parallel clines in frequency across the North American and Australian continents. This study reports that the cline is stable over at least a five-year period and that the polymorphism also demonstrates temporal shifts in allele frequency concurrent with seasonal change. The alleles were tested for effects on levels of insulin signaling, fecundity, development time, body size, stress tolerance, and lifespan. The alleles are associated with predictable differences in these traits, consistent with patterns of Drosophila life history variation across geography that likely reflect adaptation to the heterogeneous climatic environment. These results implicate insulin signaling as a major mediator of life history adaptation in Drosophila, and suggest that life history tradeoffs can be explained by extensive pleiotropy at a single locus (Paaby, 2014).


Insulin receptor pathway in C. elegans

In mammals, insulin signalling regulates glucose transport together with the expression and activity of various metabolic enzymes. In the nematode Caenorhabditis elegans, a related pathway regulates metabolism, development and longevity. Wild-type animals enter the developmentally arrested dauer stage in response to high levels of a secreted pheromone, accumulating large amounts of fat in their intestines and hypodermis. Mutants in DAF-2 (a homolog of the mammalian insulin receptor) and AGE-1 (a homolog of the catalytic subunit of mammalian phosphatidylinositol 3-OH kinase) arrest development at the dauer stage. Moreover, animals bearing weak or temperature-sensitive mutations in daf-2 and age-1 can develop reproductively, but nevertheless show increased energy storage and longevity. Null mutations in daf-16 (Drosophila homolog: foxo coding for a forkhead family transcription factor target of the insulin signaling pathway) suppress the effects of mutations in daf-2 or age-1; lack of daf-16 bypasses the need for this insulin receptor-like signalling pathway. The principal role of DAF-2/AGE-1 signalling is thus to antagonize DAF-16. daf-16 is widely expressed and encodes three members of the Fork head family of transcription factors. The DAF-2 pathway acts synergistically with the pathway activated by a nematode TGF-beta-type signal, DAF-7, suggesting that DAF-16 cooperates with nematode SMAD proteins in regulating the transcription of key metabolic and developmental control genes. The probable human orthologs of DAF-16, FKHR and AFX, may also act downstream of insulin signalling and cooperate with TGF-beta effectors in mediating metabolic regulation. These genes may be dysregulated in diabetes (Ogg, 1997).

The wild-type Caenorhabditis elegans nematode ages rapidly, undergoing development, senescence, and death in less than 3 weeks. In contrast, mutants with reduced activity of the gene daf-2, a homolog of the insulin and insulin-like growth factor receptors, age more slowly than normal and live more than twice as long. These mutants are active and fully fertile and have normal metabolic rates. The life-span extension caused by daf-2 mutations requires the activity of the gene daf-16. daf-16 appears to play a unique role in life-span regulation and encodes a member of the hepatocyte nuclear factor 3 (HNF-3)/forkhead family of transcriptional regulators. In humans, insulin down-regulates the expression of certain genes by antagonizing the activity of HNF-3, raising the possibility that aspects of this regulatory system have been conserved (Lin, 1997).

In C. elegans, mutations that reduce the activity of an insulin-like receptor (daf-2) or a phosphatidylinositol-3-OH kinase (age-1) favor entry into the dauer state during larval development and extend lifespan in adults. Downregulation of this pathway activates a forkhead transcription factor (daf-16), which may regulate targets that promote dauer formation in larvae and stress resistance and longevity in adults. In yeast, the SIR2 gene determines the lifespan of mother cells, and adding an extra copy of SIR2 extends lifespan. Sir2 mediates chromatin silencing through a histone deacetylase activity that depends on NAD (nicotinamide adenine dinucleotide) as a cofactor. A survey was performed of the lifespan of C. elegans strains containing duplications of chromosomal regions. A duplication containing sir-2.1-the C. elegans gene most homologous to yeast SIR2-confers a lifespan that is extended by up to 50%. Genetic analysis indicates that the sir-2.1 transgene functions upstream of daf-16 in the insulin-like signalling pathway. These findings suggest that Sir2 proteins may couple longevity to nutrient availability in many eukaryotic organisms (Tissenbaum, 2001).

The lifespan of Caenorhabditis elegans is regulated by the insulin/insulin-like growth factor (IGF)-1 receptor homolog DAF-2, which signals through a conserved phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Mutants in this pathway remain youthful and active much longer than normal animals and can live more than twice as long. This lifespan extension requires DAF-16, a forkhead/winged-helix transcription factor. DAF-16 is thought to be the main target of the DAF-2 pathway. Insulin/IGF-1 signaling is thought to lead to phosphorylation of DAF-16 by AKT activity, which in turn shortens lifespan. The DAF-2 pathway prevents DAF-16 accumulation in nuclei. Disrupting Akt-consensus phosphorylation sites in DAF-16 causes nuclear accumulation in wild-type animals, but, surprisingly, has little effect on lifespan. Thus the DAF-2 pathway must have additional outputs. Lifespan in C. elegans can be extended by perturbing sensory neurons or germ cells. In both cases, lifespan extension requires DAF-16. Both sensory neurons and germline activity regulate DAF-16 accumulation in nuclei, but the nuclear localization patterns are different. Together these findings reveal unexpected complexity in the DAF-16-dependent pathways that regulate aging (Lin, 2001).

Aging and limited life span are fundamental biological phenomena observed in a variety of species. Approximately 55 genes have been identified that can extend longevity when altered in Caenorhabditis elegans. These genes include an insulin-like receptor (daf-2) and a phosphatidylinositol 3-OH kinase (age-1) regulating a forkhead transcription factor (daf-16), as well as genes mediating metabolic throughput, sensory perception, and reproduction. Moreover, these mutant alleles both extend life span and increase resistance to ultraviolet (UV) radiation, heat, and oxidative stress, though the stress resistance of clk-1 is controversial. With the exception of old-1 and perhaps some other genes, all of the life-extension alleles are hypomorphic or nullomorphic. OLD-1 transmembrane tyrosine kinase (formerly TKR-1) is expressed in a variety of tissues, is stress inducible, and is a positive regulator of longevity and stress resistance. The transcription of old-1 is upregulated in long-lived age-1 and daf-2 mutants and is upregulated in response to heat, UV light, and starvation. Both RT-PCR and analysis of an OLD-1::GFP tag suggest that old-1 expression is dependent on daf-16. Importantly, old-1 is required for the life extension of age-1 and daf-2 mutants. This study reveals a new system for specifying longevity and stress resistance and suggests possible mechanisms for mediating life extension by dietary restriction and hormesis (Murakami, 2001).

The daf-2 insulin-like receptor pathway regulates development and life-span in Caenorhabditis elegans. Reduced DAF-2 signaling leads to changes in downstream targets via the daf-16 gene, a fork-head transcription factor that is regulated by DAF-2, and results in extended life-span. This study describes the first identification of genes whose expression is controlled by the DAF-2 signaling cascade. dao-1, dao-2, dao-3, dao-4, dao-8 and dao-9 are down-regulated in daf-2 mutant adults compared to wild-type adults, whereas dao-5, dao-6 and dao-7 are up-regulated. The latter genes are negatively regulated by DAF-2 signaling and positively regulated by DAF-16. Positive regulation by DAF-2 of dao-1, dao-4 and dao-8 is mediated by DAF-16, whereas daf-16 mediates only part of DAF-2 signaling for dao-2 and dao-9. Regulation by DAF-2 is most likely DAF-16 independent for dao-3 and hsp-90. RNA levels of dao-5 and dao-6 show elevated expression in daf-2 adults, as well as being strongly expressed in dauer larvae. In contrast, hsp-90 transcript levels are low in daf-2 mutant adults though they are enriched in dauer larvae, indicating overlapping but not identical mechanisms of efficient life maintenance in stress-resistant dauer larvae and long-lived daf-2 mutant adults. dao-1, dao-8 and dao-9 are homologs of the FK506 binding proteins that interact with the mammalian insulin pathway. dao-3 encodes a putative methylenetetrahydrofolate dehydrogenase. DAO-5 shows 33% identity with human nucleolar phosphoprotein P130. dao-7 is similar to the mammalian ZFP36 protein. Distinct regulatory patterns of dao genes implicate their diverse positions within the signaling network of DAF-2 pathway, and suggest they have unique contributions to development, metabolism and longevity (Yu, 2001).

C. elegans insulin-like signaling regulates metabolism, development, and life span. This signaling pathway negatively regulates the activity of the forkhead transcription factor DAF-16. daf-16 encodes multiple isoforms that are expressed in distinct tissue types and are probable orthologs of human FKHRL1, FKHR, and AFX. Human FKHRL1 can partially replace DAF-16, proving the orthology. In mammalian cells, insulin and insulin-like growth factor signaling activate AKT/PKB kinase to negatively regulate the nuclear localization of DAF-16 homologs. The absence of AKT consensus sites on DAF-16 is sufficient to cause dauer arrest in daf-2 plus animals, proving that daf-16 is the major output of insulin signaling in C. elegans. FKHR, FKRHL1, and AFX may similarly be the major outputs of mammalian insulin signaling. daf-2 insulin signaling, via AKT kinases, negatively regulates DAF-16 by controlling its nuclear localization. Surprisingly, daf-7 TGF-beta signaling also regulates DAF-16 nuclear localization specifically at the time when the animal makes the commitment between diapause and reproductive development. daf-16 function is supported by the combined action of two distinct promoter/enhancer elements, whereas the coding sequences of two major DAF-16 isoforms are interchangeable. Together, these observations suggest that the combined effects of transcriptional and posttranslational regulation of daf-16 transduce insulin-like signals in C. elegans and perhaps more generally (Lee, 2001).

Signaling from the DAF-2/insulin receptor to the DAF-16/FOXO transcription factor controls longevity, metabolism, and development in disparate phyla. To identify genes that mediate the conserved biological outputs of daf-2/insulin-like signaling, comparative genomics were used to identify 17 orthologous genes from Caenorhabditis and Drosophila, each of which bears a DAF-16 binding site in the promoter region. One-third of these DAF-16 downstream candidate genes are regulated by daf-2/insulin-like signaling in C. elegans, and RNA interference inactivation of the candidates show that many of these genes mediate distinct aspects of daf-16 function, including longevity, metabolism, and development (Lee, 2003).

let-502 rho-binding kinase and mel-11 myosin phosphatase regulate Caenorhabditis elegans embryonic morphogenesis. Genetic analysis presented here establishes the following modes of let-502 action: (1) loss of only maternal let-502 results in abnormal early cleavages, (2) loss of both zygotic and maternal let-502 causes elongation defects, and (3) loss of only zygotic let-502 results in sterility. The morphogenetic function of let-502 and mel-11 is apparently redundant with another pathway since elimination of these two genes results in progeny that undergo near-normal elongation. Triple mutant analysis indicates that unc-73 (Rho/Rac guanine exchange factor) and mlc-4 (myosin light chain) act in parallel to or downstream of let-502/mel-11. In contrast mig-2 (Rho/Rac), daf-2 (insulin receptor), and age-1 (PI3 kinase) act within the let-502/mel-11 pathway. Mutations in the sex-determination gene fem-2, which encodes a PP2c phosphatase (unrelated to the MEL-11 phosphatase), enhances mutations of let-502 and suppressed those of mel-11. fem-2's elongation function appears to be independent of its role in sexual identity since the sex-determination genes fem-1, fem-3, tra-1, and tra-3 have no effect on mel-11 or let-502. By itself, fem-2 affects morphogenesis with low penetrance. fem-2 blocks the near-normal elongation of let-502; mel-11, indicating that fem-2 acts in a parallel elongation pathway. The action of two redundant pathways likely ensures accurate elongation of the C. elegans embryo (Piekny, 2002).

In Caenorhabditis elegans, an insulin-like signaling pathway, which includes the daf-2 and age-1 genes, controls longevity and stress resistance. Downregulation of this pathway activates the forkhead transcription factor DAF-16, whose transcriptional targets are suggested to play an essential role in controlling the phenotypes governed by this pathway. The genes that have the DAF-16 consensus binding element (DBE) within putative regulatory regions have been surveyed. One such gene, termed scl-1, is a positive regulator of longevity and stress resistance. Expression of scl-1 is upregulated in long-lived daf-2 and age-1 mutants and is undetectable in a short-lived daf-16 mutant. SCL-1 is a putative secretory protein with an SCP domain and is homologous to the mammalian cysteine-rich secretory protein (CRISP) family. scl-1 is required for the extension of the life span of daf-2 and age-1 mutants, and downregulation of scl-1 reduces both life span and stress resistance of this animal. SCL-1, whose expression is dependent on DAF-16, is the first example of a putative secretory protein that positively regulates longevity and stress resistance (Ookuma, 2003).

The life span of C. elegans is extended by mutations that inhibit the function of sensory neurons. In this study, specific subsets of sensory neurons are shown to influence longevity. Certain gustatory neurons inhibit longevity, whereas others promote longevity, most likely by influencing insulin/IGF-1 signaling. Olfactory neurons also influence life span, and they act in a distinct pathway that involves the reproductive system. In addition, a putative chemosensory G protein-coupled receptor expressed in some of these sensory neurons inhibits longevity. Together these findings imply that the life span of C. elegans is regulated by environmental cues and that these cues are perceived and integrated in a complex and sophisticated fashion by specific chemosensory neurons (Alcedo, 2004).

These findings suggest that gustatory neurons are likely to influence life span by perturbing the insulin/IGF-1 pathway. One possibility is that these neurons sense cues that regulate the release of insulin/IGF-1-like hormones that influence the insulin/IGF-1 receptor DAF-2 activity. The C. elegans genome contains more than 30 insulin/IGF-1 homologs, and several of these are expressed in gustatory neurons. The model is favored that longevity-inhibiting ASI and ASG neurons exert their effects on life span by inhibiting the activities of the ASJ and ASK neurons. Thus, one possibility is that the ASI and ASG neurons prevent the longevity-promoting ASJ and ASK neurons from producing a DAF-2 antagonist. It is intriguing that double mutants that have defects in proteins thought to be required for neuronal insulin secretion and in daf-2 activity have an intermediate life span between those of the corresponding neurosecretory single mutants and daf-2 hypomorphic single mutants. This finding is consistent with the idea that some sensory neurons might secrete DAF-2 antagonists (Alcedo, 2004).

A number of insulin-like peptides have now been implicated in the regulation of aging. One such candidate DAF-2 antagonist is ins-1, but this gene is expressed not only in longevity-promoting neurons but also in longevity-inhibiting neurons. At this point, the information available about specific insulin-like peptides does not suggest simple models that explain the data. However, this may change as more is learned about the functions of these proteins. For example, it is possible that other insulin/IGF-1-like peptides function as antagonists in the ASJ and ASK neurons but not in the ASI and ASG neurons, since other insulin/IGF-1-like peptides are expressed in ASJ (Alcedo, 2004).

These observations suggest that olfactory neurons act in a regulatory pathway distinct from gustatory neurons to affect life span. (1) The combined ablation of the gustatory ASI and olfactory AWA and AWC neurons increases life span more than does ablation of either ASI or of AWA and AWC neurons alone. (2) Killing ASJ and ASK suppresses the longevity of ASI-ablated animals but not that of olfactory neuron-ablated animals. (3) The life span extension produced by killing gustatory neurons is completely daf-16 dependent, whereas the life span extension produced by killing olfactory neurons is only partially daf-16 dependent (Alcedo, 2004).

Olfactory neurons may influence life span by perturbing an endocrine signaling pathway that involves the reproductive system. Previous findings have suggested that the germline of C. elegans generates a signal that inhibits longevity and is counterbalanced by a signal from the somatic gonad that promotes longevity. Like the olfactory neurons characterized, the somatic gonad of C. elegans affects life span, at least in part, in a daf-16-independent fashion. In addition, olfactory neurons are required for the somatic gonad to influence life span. In wild-type animals, killing the somatic gonad precursors completely prevents germline ablation from extending life span, but in animals lacking olfactory neurons, it does not. One possibility is that olfactory neurons regulate the release of a hormone that allows the somatic gonad to influence longevity. If this model is correct, then it implies that, under some environmental conditions, the somatic gonad signal is silenced and may no longer be able to counterbalance the signals from the animal's germline. Alternatively, these olfactory neurons could produce a longevity signal in response to a different signal from the somatic gonad. The somatic gonad appears to regulate a pathway that involves DAF-2. Thus, as with the gustatory neurons, it is possible that the olfactory neurons influence longevity by regulating the release of insulin-like peptides (Alcedo, 2004).

Why might sensory neurons influence longevity? One environmental condition, food limitation, is known to have a dramatic effect on life span in many organisms. Caloric restriction extends life span and also delays reproduction. When ample food is restored to calorically-restricted rats, they can reproduce, even at a time when the age-matched controls are post-reproductive or dead. Thus, this response to caloric restriction has obvious survival value, since it allows animals to postpone reproduction until conditions improve. Dauer formation, which is regulated, at least in part, by sensory cues, serves the same function in C. elegans -- it allows animals to postpone reproduction under harsh environmental conditions. No obvious changes were observed in the timing of reproduction in the neuron-ablated animals; however, it is possible that the environmental cues that influence the activities of these neurons in nature also influence other neurons that control reproduction. In this way, sensory cues could affect life span and reproduction coordinately. Alternatively, certain environmental conditions could favor a shorter post-reproductive life span to prevent the aging animals from competing for resources with their progeny. A population of worms that lacks parental competition for resources should, over time, develop a significant advantage relative to populations in which such competition takes place (Alcedo, 2004).

The odr-10 gene encodes an olfactory G protein-coupled receptor that senses diacetyl, an odorant sensed by AWA neurons. odr-10 null mutants are not long-lived, implying that neither diacetyl nor its receptor regulates life span. In contrast, decreasing the mRNA levels of the putative chemosensory G protein-coupled receptor str-2, through RNA-mediated interference, extends life span. This suggests that C. elegans' life span is influenced by its perception of an environmental cue -- as yet unidentified -- that is sensed by STR-2. The identification of sensory cues that influence life span, such as those sensed by STR-2, should make it possible to address this interesting question experimentally (Alcedo, 2004).

Genetic analysis has shown that dos/soc-1/Gab1 functions positively in receptor tyrosine kinase (RTK) stimulated Ras/Map kinase signaling, through the recruitment of csw/ptp-2/Shp2. Using sensitised assays in C. elegans for let-23/Egfr and daf-2/InsR (Insulin receptor-like) signaling, it has been shown that soc-1/Gab1 inhibits phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3'-kinase (PI3K) mediated signaling. Furthermore, as well as stimulating Ras/Map kinase signaling, soc-1/Gab1 stimulates a poorly defined signaling pathway that represses class 2 daf-2 phenotypes. In addition, it is shown that SOC-1 binds the C-terminal SH3 domain of SEM-5. This binding is likely to be functional because the sem-5(n2195)G201R mutation, which disrupts SOC-1 binding, behaves in a qualitatively similar manner to a soc-1 null allele in all assays for let-23/Egfr and daf-2/InsR signaling examined. Further genetic analysis suggests that ptp-2/Shp2 mediates the negative function of soc-1/Gab1 in PI3K mediated signaling, as well as the positive function in Ras/Map kinase signaling. Other effectors of soc-1/Gab1 are likely to inhibit PLCgamma mediated signaling and stimulate the poorly defined signaling pathway that represses class 2 daf-2 phenotypes. Thus, the recruitment of soc-1/Gab1, and its effectors, into the RTK signaling complex modifies the cellular response by enhancing Ras/Map kinase signaling while inhibiting PI3K and PLCgamma mediated signaling (Hopper, 2006).

Development is typically studied as a continuous process under laboratory conditions, but wild animals often develop in variable and stressful environments. C. elegans larvae hatch in a developmentally arrested state (L1 arrest) and initiate post-embryonic development only in the presence of food (E. coli in lab). In contrast to the well-studied dauer arrest, L1 arrest occurs without morphological modification, although larvae in L1 arrest are more resistant to environmental stress than developing larvae. Consistent with its role in dauer formation and aging, insulin/insulin-like growth factor (IGF) signaling is shown to regulate L1 arrest. daf-2 insulin/IGF receptor mutants have a constitutive-L1-arrest phenotype when fed and extended survival of L1 arrest when starved. Conversely, daf-16/FOXO mutants have a defective-arrest phenotype, failing to arrest development and dying rapidly when starved. DAF-16 is required for transcription of the cyclin-dependent kinase inhibitor cki-1 in stem cells in response to starvation, accounting for the failure of daf-16/FOXO mutants to arrest cell division during L1 arrest. Other developmental events such as cell migration, cell fusion, and expression of the microRNA lin-4, a temporal regulator of post-embryonic development, are also observed in starved daf-16/FOXO mutants. These results suggest that DAF-16/FOXO promotes developmental arrest via transcriptional regulation of numerous target genes that control various aspects of development (Baugh, 2006).

Genetic and RNA interference (RNAi) screens for life span regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in Caenorhabditis elegans longevity. This pathway converges on the DAF-16 transcription factor and may regulate life span by controlling the expression of a large number of genes, including free-radical detoxifying genes, stress resistance genes, and pathogen resistance genes. A genome-wide RNAi screen was conducted to identify genes necessary for the extended life span of daf-2 mutants and ~200 gene inactivations were identified that shorten daf-2 life span. Some of these gene inactivations dramatically shorten daf-2 mutant life span but less dramatically shorten daf-2; daf-16 mutant or wild-type life span. Molecular and behavioral markers for normal aging and for extended life span in low insulin/IGF1 (insulin-like growth factor 1) signaling were assayed to distinguish accelerated aging from general sickness and to examine age-related phenotypes. Detailed demographic analysis, molecular markers of aging, and insulin signaling mutant test strains were used to filter progeric gene inactivations for specific acceleration of aging. Highly represented in the genes that mediate life span extension in the daf-2 mutant are components of endocytotic trafficking of membrane proteins to lysosomes. These gene inactivations disrupt the increased expression of the DAF-16 downstream gene superoxide dismutase sod-3 in a daf-2 mutant, suggesting trafficking between the insulin-like receptor and DAF-16. The activities of these genes may normally decline during aging (Samuelson, 2007).

Neural circuits detect environmental changes and drive behavior. The routes of information flow through dense neural networks are dynamic, but the mechanisms underlying this circuit flexibility are poorly understood. This study defined a sensory context-dependent and neuropeptide-regulated switch in the composition of a C. elegans salt sensory circuit. The primary salt detectors, ASE sensory neurons, used BLI-4 endoprotease-dependent cleavage to release the insulin-like peptide INS-6 in response to large, but not small, changes in external salt stimuli. Insulins, signaling through the insulin receptor DAF-2, functionally switched the AWC olfactory sensory neuron into an interneuron in the salt circuit. Worms with disrupted insulin signaling had deficits in salt attraction, suggesting that peptidergic signaling potentiates responses to high salt stimuli, which may promote ion homeostasis. These results indicate that sensory context and neuropeptide signaling modify neural networks and suggest general mechanisms for generating flexible behavioral outputs by modulating neural circuit composition (Leinwand, 2013).

A Caenorhabditis elegans developmental decision requires insulin signaling-mediated neuron-intestine communication

Adverse environmental conditions trigger C. elegans larvae to activate an alternative developmental program, termed dauer diapause, which renders them stress resistant. High-level insulin signaling prevents constitutive dauer formation. However, it is not fully understood how animals assess conditions to choose the optimal developmental program. This study shows that insulin-like peptide (ILP)-mediated neuron-intestine communication plays a role in this developmental decision. Consistent with, and extending, previous findings, it was shown that the simultaneous removal of INS-4, INS-6 and DAF-28 leads to fully penetrant constitutive dauer formation, whereas the removal of INS-1 and INS-18 significantly inhibits constitutive dauer formation. These ligands are processed by the proprotein convertases PC1/KPC-1 and/or PC2/EGL-3. The agonistic and antagonistic ligands are expressed by, and function in, neurons to prevent or promote dauer formation. By contrast, the insulin receptor DAF-2 and its effector, the FOXO transcription factor DAF-16, function solely in the intestine to regulate the decision to enter diapause. These results suggest that the nervous system normally establishes an agonistic ILP-dominant paradigm to inhibit intestinal DAF-16 activation and allow reproductive development. Under adverse conditions, a switch in the agonistic-antagonistic ILP balance activates intestinal DAF-16, which commits animals to diapause (Hung, 2014).

DAF-2 and ERK Couple Nutrient Availability to Meiotic Progression during Caenorhabditis elegans Oogenesis

Coupling the production of mature gametes and fertilized zygotes to favorable nutritional conditions improves reproductive success. In invertebrates, the proliferation of female germline stem cells is regulated by nutritional status. However, in mammals, the number of female germline stem cells is set early in development, with oocytes progressing through meiosis later in life. Mechanisms that couple later steps of oogenesis to environmental conditions remain largely undefined. This study shows that, in the presence of food, the DAF-2 insulin-like receptor signals through the RAS-ERK pathway to drive meiotic prophase I progression and oogenesis; in the absence of food, the resultant inactivation of insulin-like signaling leads to downregulation of the RAS-ERK pathway, and oogenesis is stalled. Thus, the insulin-like signaling pathway couples nutrient sensing to meiotic I progression and oocyte production in C. elegans, ensuring that oocytes are only produced under conditions favorable for the survival of the resulting zygotes (Lopez, 2013).

Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans

Inhibition of DAF-2 (insulin-like growth factor 1 [IGF-1] receptor) or RSKS-1 (S6K), key molecules in the insulin/IGF-1 signaling (IIS) and target of rapamycin (TOR) pathways, respectively, extend lifespan in Caenorhabditis elegans. However, it has not been clear how and in which tissues they interact with each other to modulate longevity. This study demonstrates that a combination of mutations in daf-2 and rsks-1 produces a nearly 5-fold increase in longevity that is much greater than the sum of single mutations. This synergistic lifespan extension requires positive feedback regulation of DAF-16 (FOXO) via the AMP-activated protein kinase (AMPK) complex. Furthermore, germline was identified as the key tissue for this synergistic longevity. Moreover, germline-specific inhibition of rsks-1 activates DAF-16 in the intestine. Together, these findings highlight the importance of the germline in the significantly increased longevity produced by daf-2 rsks-1, which has important implications for interactions between the two major conserved longevity pathways in more complex organisms (Chen, 2013).

IIS and TOR pathways play conserved roles in modulating lifespan in multiple species. However, it is unclear how they might interactively modulate aging. This study set out to address this question by constructing a daf-2 rsks-1 double mutant, which has reduced function of IIS and an important branch of the TOR pathway. Surprisingly, the daf-2 rsks-1 double mutant showed a nearly 5-fold lifespan extension. This phenotype is defined as a synergistic lifespan extension, based on the observation that the longevity of the daf-2 rsks-1 double mutant is beyond the combined effects of rsks-1 and daf-2 single mutants. This synergistic longevity phenotype cannot be explained by the hypothesis that daf-2 and rsks-1 function in parallel to modulate lifespan independently, since an additive effect would be expected under such an assumption (Chen, 2013).

The synergistic longevity phenotype is different from what we previously reported; i.e., that rsks-1 RNAi further extended the daf-2 lifespan by 24%. One major difference in the experimental procedures used is that in the previous study, daf-2 animals were treated with rsks-1 RNAi only during adulthood, whereas in the current work, a double mutant was made that carries the putative null allele of rsks-1 throughout life (Chen, 2013).

When daf-2 animals were treated with rsks-1 RNAi for two generations, resulting in a more complete reduction in rsks-1 mRNA levels, a 54% further lifespan extension was observed. These results suggest that inhibition of rsks-1 during development is critical for the synergistic longevity phenotype. Consistently, inhibition of the RSKS-1 upstream activator LET-363/CeTOR in daf-2 during adulthood led to a 17% additive lifespan extension. Since let-363 is an essential gene, inhibition of which during development leads to larval arrest, a pharmaceutical approach was used to inhibit let-363 by treating animals with rapamycin. Rapamycin treatment throughout life extended the lifespan of N2 and daf-2 animals by 26% and 45%, respectively. There are multiple possible reasons why rapamycin treatment could not extend the lifespan of daf-2 animals as much as the rsks-1 deletion mutant did. One possibility is that rapamycin treatment did not fully block RSKS-1, which is required for the synergistic longevity. Another possibility is that since rapamycin treatment at this dosage has been shown to inhibit both TOR complex 1 and complex 2 activities (Robida-Stubbs et al., 2012), the drug might also affect other lifespan-determinant genes. Nevertheless, these results are consistent with the idea that inhibiting rsks-1 in daf-2 during development leads to a synergistic lifespan extension (Chen, 2013).

Previous studies showed that null mutants of age-1, which encodes a catalytic subunit of the phosphatidylinositol-3-kinase (PI3K) in the IIS pathway, exhibit an exceptional lifespan extension in a DAF-16-dependent manner (Chen, 2013).

Since the daf-2 mutations that were used in this study are not null alleles, one possible explanation for the synergistic longevity produced by daf-2 rsks-1 is that the rsks-1 deletion makes daf-2 mutant phenotypes more severe. This is unlikely to be true, because many aging-related phenotypes of daf-2 are not enhanced by the rsks-1 deletion.rsks-1 does not affect daf-2-mediated dauer arrest, and rsks-1 has a minor or even opposite effect on most stress resistance. Understanding why these phenotypes are uncoupled from the synergistically prolonged longevity produced by daf-2 rsks-1 will help to elucidate the basic mechanisms of aging (Chen, 2013).

TOR plays a conserved role in dietary restriction (DR)-mediated lifespan extension. The effect of nutrients on the synergistic longevity was tested using the DR-FD regimen (FD stands for food deprivation). The rsks-1 single mutant did not show a lifespan extension under DR, which is consistent with the idea that DR and reduced TOR signaling function through overlapping mechanisms to extend lifespan. Interestingly, the synergistic longevity produced by daf-2 rsks-1 is nutrient independent, suggesting that rsks-1 functions through unidentified mechanisms to further extend the lifespan of daf-2 animals (Chen, 2013).

To better understand the molecular mechanisms of the synergistic longevity produced by daf-2 rsks-1, this study set out to identify critical mediators by testing known regulators of IIS or rsks-1. Heat-shock factor 1 (HSF-1) is critical for daf-2-mediated lifespan extension. Inhibition of hsf-1 almost completely abolished the lifespan extension produced by daf-2 rsks-1. Lifespan extension via genetic or pharmaceutical inhibition of TOR requires the IIS downstream transcription factor SKN-1. Surprisingly, inhibition of skn-1 by RNAi had little effect on the synergistic longevity produced by daf-2 rsks-1. Similarly, inhibition of PHA-4, a FOXA transcription factor that is required for the rsks-1 single mutant-mediated lifespan extension, did not affect the lifespan of daf-2 rsks-1. This is further evidence that the mechanism of the synergistic longevity in the daf-2 rsks-1 double mutant is distinct from the lifespan extension caused by the single mutants (Chen, 2013).

Microarray studies were performed and genes were identified that are differentially expressed in daf-2 rsks-1. A genetic screen using RNAi helped identify the AMPK complex (see Drosophila AMP-activated protein kinase alpha subunit) as the key mediator of the synergistic longevity produced by daf-2 rsks-1. Quantitative analysis of the lifespan data indicated that suppression of the daf-2 rsks-1 lifespan by inhibition of AMPK was not due to general sickness. Instead, inhibition of AMPK suppressed the synergy part of the lifespan extension. Further analysis identified a positive feedback regulation of DAF-16 via AMPK in the daf-2 rsks-1 mutant. AMPK plays important roles in various cellular functions. Under energy-starved conditions, AMPK is activated to promote catabolism and thus ATP production. Further characterization of the role of AMPK in metabolism will enhance understanding of the synergistic longevity produced by daf-2 rsks-1 (Chen, 2013).

Both IIS and signals from the reproductive system have endocrine functions. Modulation of these pathways in one tissue leads to nonautonomous activation of DAF-16 in the intestine. To better understand how aging is coordinately modulated across multiple tissues, the involvement of key regulators of the daf-2 rsks-1-mediated synergistic longevity were tested by tissue-specific RNAi. It was found that rsks-1, daf-16, and aak-2 function in the germline to regulate the synergistic lifespan extension, which can also be suppressed by a genetic mutation that causes germline overproliferation or by inhibition of key mediators of germline signaling. In addition, inhibiting rsks-1 in the germline leads to nonautonomous activation of DAF-16 in the intestine. Previous studies on the tissue-specific requirements of key longevity determinants, including DAF-16, mainly employed transgenic rescue approaches. However, the traditional microinjection method creates transgenic lines with a high copy number of transgenes, which will be silenced in the germline. The results indicate that the germline is an important tissue for integrating signals from the IIS pathway and S6K for lifespan determination (Chen, 2013).

Similar to the rsks-1 single mutant, daf-2 rsks-1 animals showed significantly delayed, prolonged, and overall reduced reproduction. This is consistent with a recent study showing that RSKS-1 acts in parallel with the IIS pathway to play an essential role in establishing the germline stem cell/progenitor pool. Interestingly, RSKS-1 functions cell autonomously to regulate establishment of the germline progenitor. This effect is independent of its known suppressors in the regulation of lifespan. These findings suggest that the synergistic longevity of daf-2 rsks-1 cannot simply be linked with its functions in germline development and reproduction (Chen, 2013).

In C. elegans, the intestine carries out multiple nutrient-related functions and is the site for food digestion and absorption, fat storage, and immune response. DAF-16 is one of the essential transcription factors that function in the intestine to modulate lifespan. It was found that intestinal-specific inhibition of daf-16, aak-2, or hsf-1 largely abolishes the synergistic lifespan extension of daf-2 rsks-1. However, knockdown of rsks-1 in the intestine only has an additive effect on daf-2 lifespan, suggesting that rsks-1 may function through nonautonomous mechanisms to activate DAF-16 (Chen, 2013).

The hypodermis is considered as part of the epithelial system in C. elegans. It is involved in basic body plan establishment, cell fate specification, axon migration, apoptotic cells removal, and fat storage. Hypodermis-specific knockdown of rsks-1 in daf-2 also leads to synergistic lifespan extension, and that hypodermis-specific knockdown of daf-16 significantly reduces the synergistic lifespan extension. These results provide evidence for the important role of the hypodermis in lifespan determination. In future studies, it will be interesting to examine which biological functions of the hypodermis are involved in regulating the synergistic longevity by daf-2 rsks-1 (Chen, 2013).

Previous studies showed that muscle decline is one of the major physiological causes of aging in C. elegans. Neither rsks-1 nor the downstream regulators daf-16, hsf-1, and aak-2 seem to function in the muscle to modulate the synergistic lifespan extension. However, the possibility that these regulators may function in other tissues to nonautonomously regulate muscle functions in daf-2 rsks-1 cannot be ruled out. Characterization of age-dependent muscle decline in daf-2 rsks-1 will help to elucidate whether muscle functions are important for the synergistic lifespan extension (Chen, 2013).

There are limitations to assessing tissue-specific involvement of key regulators in lifespan determination by RNAi, such as uncertainty of knockdown efficiency and potential leakiness. It has been reported that in rrf-1 mutants, RNAi can be processed in certain somatic tissues, including the intestine, at least for the genes tested. However, the critical function of rsks-1 in the germline is unlikely to be an artifact, as rsks-1 knockdown in the intestine of daf-2 animals did not lead to synergistic lifespan extension. Moreover, inhibition of certain strong suppressors of daf-2 rsks-1 (e.g., hsf-1) in the intestine, but not in the germline, significantly decreased the synergistic lifespan extension produced by daf-2 rsks-1. Further analyses with single-copied, isoform-specific transgenic rescue will help to quantitatively determine the tissue-specific involvement of key regulators in the synergistic lifespan extension produced by daf-2 rsks-1 (Chen, 2013).

It has not been clear whether DAF-16 is quantitatively more active or is uniquely activated in certain tissues, such as the germline of daf-2 rsks-1. Although the AMPK-mediated positive feedback regulation of DAF-16 was identified based on genes that are expressed to a greater extent in daf-2 rsks-1 animals, it is speculated that the double mutant has some unique properties, as shown in dauer formation and various stress-tolerance assays. The data from the phenotypic analysis of the double mutant and epistasis analysis of tissue requirement of DAF-16 suggest that with the rsks-1 deletion, DAF-16 plays a more important role in certain tissues, such as the germline, to further extend the lifespan of daf-2. Characterization of the genes that are uniquely upregulated in daf-2 rsks-1 or those that are regulated independently of DAF-16 will help distinguish these models (Chen, 2013).

In conclusion, this study found that the daf-2 rsks-1 double mutant shows a synergistic lifespan extension, which is achieved through positive feedback regulation of DAF-16 by AMPK. Tissue- specific epistasis analysis suggests that this enhanced activation of DAF-16 is initiated by signals from the germline, and that the germline tissue may play a key role in integrating the interactions between daf-2 and rsks-1 to cause a synergistic lifespan extension. Since DAF-2, RSKS-1, AMPK, and DAF-16 are highly conserved molecules, similar regulation may also exist in mammals. Further characterization of the daf-2 rsks-1-mediated synergistic longevity will contribute to a better understanding of the molecular mechanisms of aging and age-related diseases (Chen, 2013).

Insulin signaling is involved in the regulation of worker division of labor in honey bee colonies

It has been proposed that one route of behavioral evolution involves novel regulation of conserved genes. Age-related division of labor in honey bee colonies, a highly derived behavioral system, involves the performance of different feeding-related tasks by different groups of individuals. Older bees acquire the colony's food by foraging for nectar and pollen, and the younger 'nurse' bees feed larvae processed foods. The transition from hive work to foraging has been shown to be socially regulated and associated both with decreases in abdominal lipid stores and with increases in brain expression of genes implicated in feeding behavior in Drosophila melanogaster. This study shows that division of labor is influenced by a canonical regulator of food intake and energy balance in solitary species, the insulin/insulin-like growth factor signaling (IIS) pathway. Foragers had higher levels of IIS gene expression in the brain and abdomen than did nurses, despite their low lipid stores. These differences are likely nutritionally mediated because manipulations that induced low lipid stores in young bees also up-regulated these genes. Changes in IIS also causally influenced the timing of behavioral maturation: inhibition of the insulin-related target of rapamycin pathway delayed the onset of foraging in a seasonally dependent manner. In addition, pathway analyses of microarray data revealed that nurses and foragers differ in brain energy metabolism gene expression, but the differences are opposite predictions based on their insulin-signaling status. These results suggest that changes in the regulation of the IIS pathway are associated with social behavior (Ament, 2008).

Molecular pathways that regulate hunger and food-gathering behavior in solitary species influence the age at which worker honey bees shift from working in the hive to collecting food for their colony. Therefore, the regulation of honey bee division of labor, a highly derived trait, involves widely conserved nutrient-sensing or metabolic pathways, in addition to feeding-related and nutritionally related genes (Ament, 2008).

The finding that IIS gene expression is up-regulated in the brain by low nutrient stores and in foragers (Corona, 2007) differs from commonly observed patterns of expression in other species in two ways. First, the direction of the response is reversed; high levels of nutrient stores typically lead to enhanced insulin signaling. Second, whereas AmIlp1 and AmInR1 expression are positively correlated, insulin-signaling activity down-regulates insulin receptor gene expression in Drosophila and in vertebrate cell lines by inhibiting FoxO. This feedback results in a homeostatic mechanism that ensures a rapid but brief response to nutritional changes (Ament, 2008).

The current results suggest roles for insulin signaling in the brain and fat body. Increased ilp1 production in the brain may influence behavior through local action on neuronal circuits that control foraging and also may affect non-brain targets, such as the fat bodies in the abdomen. High levels of inR1 and inR2 in the abdomen should maximize the responsiveness of abdominal tissues to circulating ILPs. However, it cannot be discerned whether the increase in insulin signaling during behavioral maturation is a cause or consequence of lipid loss. A few studies in other insect species suggest that ILPs can have catabolic functions in insects, so a causal relationship is possible. The nature of this speculative brain-abdomen communication system in bees is unknown, but similar systems are well studied in vertebrates (Ament, 2008).

It is possible that the combination of high brain ilp1 and high abdominal inR1 in foragers reflects a change in the adipostatic set point relative to nurses, rather than the traditional homeostatic mechanism associated with insulin signaling. In this view, the combination of high insulin synthesis and high insulin sensitivity maintains, or perhaps causes, a shift from high to low adiposity during behavioral maturation (and in response to experimental nutritional manipulations). Similar reasoning has been used to explain relationships between nutrient-sensing pathways and variation in nutrient stores in the contexts of mammalian torpor and insect diapause (Ament, 2008).

'Reversed' IIS gene expression and the suggested set point regulation do not occur in all contexts in honey bees. More typical homeostatic regulation is seen during larval development; ilp1 in honey bee larvae is up-regulated by good nutrition. It is not known why these differences in IIS in honey bees appear to be limited to behavioral maturation. Perhaps this is because the system of social foraging in honey bees requires that they forage when they are not personally hungry (Ament, 2008).

There were seasonal changes in IIS brain gene expression and the effects of IIS on behavioral maturation, but these changes were limited to small, not large, colonies. It is speculated that this might have been because large colonies are able to maintain more stable levels of food stores and that the seasonal effects detected in late summer in small colonies would have been detected in large colonies sampled later in the fall than was done in this study. It is possible that the use of small colonies made it easier to expose the seasonal effects of IIS in honey bee colonies (Ament, 2008).

A surprising result was that the transition from in-hive tasks to foraging was associated with a decrease in whole-brain energy metabolism gene expression that does not appear to be caused either by insulin or by JH, two hormones that have causal effects on behavioral maturation. Alternatively, insulin might regulate these changes, but in the opposite direction to other tissues and species. Perhaps high levels of brain energy metabolism are required in nurses for energy-intensive processes such as brain plasticity that are not necessarily correlated with metabolism in other tissues. Changes in brain structure occur throughout the lifespan of worker honey bees but are more intense in young bees (Ament, 2008).

Another explanation for the high levels of brain energy metabolism in nurse bees is that whole-brain analyses of energy metabolism pathways do not adequately reflect what is going on in specific brain regions. In most insect brains, ILPs are produced primarily in a small cluster of neurosecretory cells, but the distribution of insulin receptors in the bee brain is not known (Ament, 2008).

Insulin signaling influences diverse aspects of phenotypic plasticity in honey bees. Insulin signaling has been implicated in the regulation of caste (queen vs. worker) determination in honey bees, and insulin-signaling genes are among the more promising candidate genes located in quantitative trait loci associated with genetic variation for honey bee foraging behavior. Several models have been proposed to explain how insulin signaling can influence diverse aspects of phenotypic plasticity in honey bees. The current experiments confirm a specific prediction of Corona (2007) by showing that low nutrient stores can increase insulin signaling. However, the context specificity of this effect implies that interactions among insulin signaling, nutrition, JH, Vg, and the environment are more complicated than had previously been imagined (Ament, 2008).

The results support the notion that molecular pathways that govern nutritional state and feeding behavior in solitary animals represent one 'toolkit' that can be used in the evolution of division of labor in social insects. Learning how and why some components of insulin-signaling pathways are more evolutionarily labile than others will help understand the molecular basis of behavior (Ament, 2008).

An insulin-like signaling pathway mediates the environmental influence on the switch between the C. elegans developmental programs of reproductive growth versus dauer arrest. However, the specific role of endogenous insulin-like peptide (ILP) ligands in mediating the switch between these programs remains unknown. C. elegans has 40 putative insulin-like genes, many of which are expressed in sensory neurons and interneurons, raising the intriguing possibility that ILPs encode different environmental information to regulate the entry into, and exit from, dauer arrest. These two developmental switches can have different regulatory requirements: this study shows that the relative importance of three different ILPs varies between dauer entry and exit. Not only was it found that one ILP, ins-1, ensures dauer arrest under harsh environments and that two other ILPs, daf-28 and ins-6, ensure reproductive growth under good conditions, it is also shown that daf-28 and ins-6 have non-redundant functions in regulating these developmental switches. Notably, daf-28 plays a more primary role in inhibiting dauer entry, whereas ins-6 has a more significant role in promoting dauer exit. Moreover, the switch into dauer arrest surprisingly shifts ins-6 transcriptional expression from a set of dauer-inhibiting sensory neurons to a different set of neurons, where it promotes dauer exit. Together, these data suggest that specific ILPs generate precise responses to dauer-inducing cues, such as pheromones and low food levels, to control development through stimulus-regulated expression in different neurons (Cornils, 2011).

Insulin/IGF1 sgnaling inhibits age-dependent axon regeneration

The ability of injured axons to regenerate declines with age, yet the mechanisms that regulate axon regeneration in response to age are not known. This study shows that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2. DAF-2's function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO. DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron-specific, and genetically regulated process. In addition, DAF-18/PTEN was found to inhibit regeneration independently of age and FOXO signaling via the TOR pathway. Finally, DLK-1, a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and required for both DAF-16- and DAF-18-mediated regeneration. Together, these data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR (Byrne, 2004).

Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens

The short day lengths of late summer program the mosquito Culex pipiens to enter a reproductive diapause characterized by an arrest in ovarian development and the sequestration of huge fat reserves. It is suggested that insulin signaling and FOXO (forkhead transcription factor), a downstream molecule in the insulin signaling pathway, mediate the diapause response. When RNAi was used to knock down expression of the insulin receptor in nondiapausing mosquitoes (those reared under long day lengths) the primary follicles were arrested in a stage comparable to diapause. The mosquitoes could be rescued from this developmental arrest with an application of juvenile hormone, an endocrine trigger known to terminate diapause in this species. When dsRNA directed against FOXO was injected into mosquitoes programmed for diapause (reared under short day lengths) fat storage was dramatically reduced and the mosquito's lifespan was shortened, results suggesting that a shutdown of insulin signaling prompts activation of the downstream gene FOXO, leading to the diapause phenotype. Thus, the results are consistent with a role for insulin signaling in the short-day response that ultimately leads to a cessation of juvenile hormone production. The similarity of this response to that observed in the diapause of Drosophila melanogaster (Tu, 2005; Williams, 2006) and in dauer formation of Caenorhabditis elegans suggests a conserved mechanism regulating dormancy in insects and nematodes (Sim, 2008).

Insulin signaling is essential for normal growth in insects, and arguably it is the most important regulator of insect growth and size. This pathway has been implicated in diverse roles including the immune response, apoptosis, longevity, and energy metabolism. In addition, suppression of insulin signaling has been implicated in the induction of adult diapause in Drosophila and in dauer formation of the nematode C. elegans. The results of this study suggest that insulin signaling is integral to diapause in the mosquito C. pipiens as well. This common theme across taxa thus suggests a conserved role for the insulin signaling pathway for developmental and reproductive arrests among insects and other invertebrates (Sim, 2008).

The fact that methoprene, a JH analog, can counter the ovarian arrest caused by the down-regulation of Culex InR indicates that insulin signaling has a significant role mediating JH synthesis in C. pipiens. Several lines of evidence indicate that JH synthesis is shut down during diapause in C. pipiens, and experiments rescuing the double-stranded RNAi InR shutdown of development with the JH analog methoprene support a causative link between insulin signaling and JH production. The responsiveness of InR mutants in Drosophila to JH also supports such a connection. In nondiapausing mosquitoes, the corpora allata synthesize JH immediately after adult eclosion, and JH titers reach peak activity during that first week. Knocking down the InR has likely blocked JH production in these long-day females, thus generating the diapause phenotype (Sim, 2008).

In C. elegans and Drosophila, insulin signals through a conserved PI3-kinase/Akt pathway to ultimately phosphorylate the FOXO protein and block the translocation of this protein into the nucleus. Thus, suppression of the insulin signal likely causes the FOXO protein to be translocated into the nucleus to initiate transcription of its downstream genes, some of which are known to be involved in key diapause characters such as the metabolic switch toward lipid storage and protection from reactive oxygen species. The results of this study suggest that these functional roles for FOXO are evident in diapausing C. pipiens as well. Suppression of FOXO by RNAi in diapausing mosquitoes resulted in loss of two key characters essential for successful overwintering: fat hypertrophy and extended lifespan. An antioxidant role is also suggested by the results elicited by a coinjection of dsFOXO and Mn(III)TBAP, an exogenous substitute for oxidoreductase: coinjection increased the lifespan and countered the mortality observed by an injection of dsFOXO alone. This result suggests that adding the oxidoreductase function enables the mosquito to cope with the stressful conditions of food shortage and environmental stress evoked by suppression of FOXO. Down-regulating the FOXO gene possibly impairs expression of oxidoreductases or small heat-shock proteins that enhance survival during diapause. The introduction of exogenous Mn(III)TBAP may, at least partially, compensate for the function of stress-responsive proteins that may be missing in FOXO RNAi mosquitoes (Sim, 2008).

In summary, these data from C. pipiens support the hypothesis that the insulin signaling pathway and forkhead transcription factor control key characters of diapause, including the metabolic switch to lipid storage, the halt in ovarian development, and enhanced overwintering survival. It is proposed that, under long day lengths, insulin signaling leads to the production of JH needed to prompt ovarian development, and, concurrently, FOXO is suppressed, thus preventing accumulation of fat stores. By contrast, in response to short day lengths, the insulin signaling pathway is shut down, which in turn halts synthesis of the JH needed for ovarian development and releases the suppression of FOXO, leading to accumulation of lipid and the stress tolerance characteristic of diapause. The concurrence of these observations with the proposed involvement of the insulin signaling pathway in other forms of dormancy suggests a mechanism common to diverse forms of developmental arrest (Sim, 2008).

Alternative splicing of Insulin receptor

Insulin signaling is mediated by a complex network of diverging and converging pathways, with alternative proteins and isoforms at almost every step in the process. The two major pathways described to date, which employ insulin receptors as the primary target, include signaling via mitogen-activated protein (MAP) kinases and phosphoinositol-3 kinase (PI3K). The insulin receptor (IR), the first step in these cascades, exists in two isoforms as a result of alternative mRNA splicing of the 11th exon of the insulin proreceptor transcript. The respective sequence coding for 12 amino acids in the C terminus of the alpha chain of the receptor is lacking in A type (IR-A), or Ex11-, whereas it is contained in the B type (IR-B), or Ex11+. To date, no insulin-induced effect has been reported that discriminates signaling via A- and B-type receptors (Leibiger, 2001).

Insulin activates the transcription of its own gene and that of the beta cell glucokinase gene (betaGK) by different mechanisms. Whereas insulin gene transcription is promoted by signaling through insulin receptor A type, PI3K class Ia, and p70s6k, insulin stimulates the betaGK gene by signaling via insulin receptor B type), PI3K class II-like activity, and PKB (c-Akt). These data provide evidence for selectivity in insulin action via the two isoforms of the insulin receptor, the molecular basis being preferential signaling through different PI3K and protein kinases (Leibiger, 2001).

Developmental roles of insulin directed pathways

The insulin-like growth factors (IGFs) are well known mitogens, both in vivo and in vitro, while functions in cellular differentiation have also been indicated. A new role for the IGF pathway in regulating head formation has been demonstrated in Xenopus embryos. Both IGF-1 and IGF-2, along with their receptor IGF-1R, are expressed early during embryogenesis, and the IGF-1R is present particularly in anterior and dorsal structures. Overexpression of IGF-1 leads to anterior expansion of head neural tissue as well as formation of ectopic eyes and cement gland, while IGF-1 receptor depletion using antisense morpholino oligonucleotides drastically reduces head structures. Furthermore, IGF signaling exerts this effect by antagonizing the activity of the Wnt signal transduction pathway in the early embryo, at the level of ß-catenin. Thus, the IGF pathway is required for head formation during embryogenesis (Richard-Parpaillon, 2002).

Wnt signaling is involved in numerous developmental processes, such as dorsal axis formation, patterning of the central nervous system, and establishment of cell polarity. The pathway is tightly regulated during embryogenesis and it is becoming increasingly clear that crossregulation between Wnt and other signaling pathways contributes to the complexity and specificity of Wnt activity. For example, retinoid signaling and a specific MAP kinase pathway (TAK/NLK) can both inhibit Wnt activity. However, previous evidence for an interaction between the IGF and the Wnt pathways is limited. IGF-1 stimulation induces a rapid tyrosine-phosphorylation of ß-catenin in a cell line derived from a human colonic adenocarcinoma. It has also been shown that the phosphorylation of ß-catenin induced by IGF-1 leads to a dissociation of the pool of ß-catenin, which is bound to E-cadherin at the plasma membrane, resulting in its relocation to the cytoplasm (Richard-Parpaillon, 2002 and references therein).

However, despite this accumulation of cytoplasmic ß-catenin, no enhancement of Wnt activity is observed after stimulation by IGF-1 alone, as determined by using the Wnt-responsive luciferase reporter construct TOP-FLASH. Recent structural studies might provide an explanation for this paradox. It has been shown that the charged residues involved in this interaction between ß-catenin/E-cadherin are the same as those required for the ß-catenin/ TCF interaction. Thus, this raises the interesting possibility that tyrosine-phosphorylation of ß-catenin, which blocks its association with E-cadherin may also prevent interaction between this molecule and its downstream effector Tcf. This is a potential point at which IGF signaling may inhibit the Wnt pathway. In the future, it will be interesting to investigate this hypothesis further, and also to determine whether the PI3K or the MAPK activated by IGF-1R may be involved in this process (Richard-Parpaillon, 2002).

Insulin receptor signaling regulates synapse number, dendritic plasticity, and circuit function in vivo

Insulin receptor signaling has been postulated to play a role in synaptic plasticity; however, the function of the insulin receptor in CNS is not clear. To test whether insulin receptor signaling affects visual system function, light-evoked responses were recorded in optic tectal neurons in living Xenopus tadpoles. Tectal neurons transfected with dominant-negative insulin receptor (dnIR), which reduces insulin receptor phosphorylation, or morpholino against insulin receptor, which reduces total insulin receptor protein level, have significantly smaller light-evoked responses than controls. dnIR-expressing neurons have reduced synapse density as assessed by EM, decreased AMPA mEPSC frequency, and altered experience-dependent dendritic arbor structural plasticity, although synaptic vesicle release probability, assessed by paired-pulse responses, synapse maturation, assessed by AMPA/NMDA ratio and ultrastructural criteria, are unaffected by dnIR expression. These data indicate that insulin receptor signaling regulates circuit function and plasticity by controlling synapse density (Chiu, 2008).

IGF-1 activates a cilium-localized noncanonical Gbetagamma signaling pathway that regulates cell-cycle progression

Primary cilia undergo cell-cycle-dependent assembly and disassembly. Emerging data suggest that ciliary resorption is a checkpoint for S phase reentry and that the activation of phospho(T94)Tctex-1 (a dynein light-chain protein) couples these two events. However, the environmental cues and molecular mechanisms that trigger these processes remain unknown. This study shows that insulin-like growth-1 (IGF-1) accelerates G1-S progression by causing cilia to resorb. The mitogenic signals of IGF-1 are predominantly transduced through IGF-1 receptor (IGF-1R) on the cilia of fibroblasts and epithelial cells. At the base of the cilium, phosphorylated IGF-1R activates an AGS3-regulated Gβγ signaling pathway that subsequently recruits phospho(T94)Tctex-1 to the transition zone. Perturbing any component of this pathway in cortical progenitors induces premature neuronal differentiation at the expense of proliferation. These data suggest that during corticogenesis, a cilium-transduced, noncanonical IGF-1R-Gβγ-phospho(T94)Tctex-1 signaling pathway promotes the proliferation of neural progenitors through modulation of ciliary resorption and G1 length (Yeh, 2013).

Astrocytic insulin signaling couples brain glucose uptake with nutrient availability

Astrocytic insulin signaling co-regulates hypothalamic glucose sensing and systemic glucose metabolism. Postnatal ablation of insulin receptors (IRs; see Drosophila Insulin-like receptor) in glial fibrillary acidic protein (GFAP)-expressing cells affects hypothalamic astrocyte morphology, mitochondrial function, and circuit connectivity. Accordingly, astrocytic IR ablation reduces glucose-induced activation of hypothalamic pro-opio-melanocortin (POMC) neurons and impairs physiological responses to changes in glucose availability. Hypothalamus-specific knockout of astrocytic IRs, as well as postnatal ablation by targeting glutamate aspartate transporter (GLAST)-expressing cells, replicates such alterations. A normal response to altering directly CNS glucose levels in mice lacking astrocytic IRs indicates a role in glucose transport across the blood-brain barrier (BBB). This was confirmed in vivo in GFAP-IR KO mice by using positron emission tomography and glucose monitoring in cerebral spinal fluid. It is concluded that insulin signaling in hypothalamic astrocytes co-controls CNS glucose sensing and systemic glucose metabolism via regulation of glucose uptake across the BBB (Garcia-Caceres, 2016).

Insulin receptor and Diabetes

Peripheral insulin resistance and impaired insulin action are the primary characteristics of type 2 diabetes. The first observable defect in this major disorder occurs in muscle, where glucose disposal in response to insulin is impaired. Insulin-like growth factor-I (IGF-I) and insulin are closely similar in both structure and function, and both can stimulate glucose uptake in muscle. The IGF-I receptor (IGF-IR) represents another potential target to develop useful animal models for human diabetes. A transgenic mouse has been developed with a dominant-negative insulin-like growth factor-I receptor (KR-IGF-IR) specifically targeted to the skeletal muscle. Expression of KR-IGF-IR results in the formation of hybrid receptors between the mutant and the endogenous IGF-I and insulin receptors, thereby abrogating the normal function of these receptors and leading to insulin resistance. Pancreatic ß-cell dysfunction develops at a relative early age, resulting in diabetes. These mice provide an excellent model to study the molecular mechanisms underlying the development of human type 2 diabetes (Fernandez, 2001).


Search PubMed for articles about Drosophila Insulin-like receptor

Alcedo, J. and Kenyon, C. (2004). Regulation of C. elegans longevity by specific gustatory and olfactory neurons. Neuron 41: 45-55. 14715134

Ament, S. A., Corona, M., Pollock, H. S. and Robinson, G. E. (2008). Insulin signaling is involved in the regulation of worker division of labor in honey bee colonies. Proc. Natl. Acad. Sci. 105(11) :4226-31. PubMed Citation: 18337502

Amoyel, M., Hillion, K. H., Margolis, S. R. and Bach, E. A. (2016). Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues. Development 143(21):3914-3925 PubMed ID: 27633989

Araki, E., et al. (1994). Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372: 186-190. 7526222

Avet-Rochex, A., Kaul, A. K., Gatt, A. P., McNeill, H. and Bateman, J. M. (2012). Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain. Development 139(15): 2763-72. PubMed Citation: 22745312

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

Baugh, L. R. and Sternberg, P. W. (2006). DAF-16/FOXO regulates transcription of cki-1/Cip/Kip and repression of lin-4 during C. elegans L1 arrest. Curr. Biol. 16(8): 780-5. 16631585

Belgacem, Y. H. and Martin, J. R. (2006). Disruption of insulin pathways alters trehalose level and abolishes sexual dimorphism in locomotor activity in Drosophila. J. Neurobiol. 66(1): 19-32. PubMed Citation: 16187303

Paaby, A. B., Bergland, A. O., Behrman, E. L. and Schmidt, P. S. (2014). A highly pleiotropic amino acid polymorphism in the Drosophila insulin receptor contributes to life history adaptation. Evolution 68(12):3395-409. PubMed ID: 25319083

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. Current Biology 11: 213-221. 11250149

Butler, A. A. and Roith, D. L. (2001). Control of growth by the somatropic axis: Growth hormone and the insulin-like growth factors have related and independent roles. Annu. Rev. Physiol. 63: 141-164. 11181952

Byrne, A. B., Walradt, T., Gardner, K. E., Hubbert, A., Reinke, V. and Hammarlund, M. (2014). Insulin/IGF1 sgnaling inhibits age-dependent axon regeneration. Neuron 81: 561-573. PubMed ID: 24440228

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, C., Jack, J. and Garofalo, R. S. (1996). The Drosophila insulin receptor is required for normal growth. Endocrinology 137(3): 846-856. 8603594

Chen, D., Li, P. W., Goldstein, B. A., Cai, W., Thomas, E. L., Chen, F., Hubbard, A. E., Melov, S. and Kapahi, P. (2013). Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans. Cell Rep 5: 1600-1610. PubMed ID: 24332851; Graphical Abstract

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

Chiu, S. L., Chen, C. M. and Cline, H. T. (2008). Insulin receptor signaling regulates synapse number, dendritic plasticity, and circuit function in vivo. Neuron 58(5): 708-19. PubMed Citation: 18549783

Clancy, D. J., et al. (2001). Extension of life-span by loss of Chico, a Drosophila Insulin receptor substrate protein. Science 292: 104-6. 11292874

Choi, N. H., Lucchetta, E. and Ohlstein B. (2011). Nonautonomous regulation of Drosophila midgut stem cell proliferation by the insulin-signaling pathway. Proc. Natl. Acad. Sci. 108(46): 18702-7. PubMed Citation: 22049341

Cornils, A., Gloeck, M., Chen, Z., Zhang, Y. and Alcedo, J. (2011). Specific insulin-like peptides encode sensory information to regulate distinct developmental processes. Development 138(6): 1183-93. PubMed Citation: 21343369

Corona, M., et al. (2007). Vitellogenin, juvenile hormone, insulin signaling, and queen honey bee longevity. Proc. Natl. Acad. Sci. 104: 7128-7133. PubMed Citation: 17438290

Danielsen, A. G., et al. (1995). Activation of protein kinase Ca inhibits signaling by members of the insulin receptor family. J. Biol. Chem. 270(37): 21600-21605. 7545165

Demontis, F. and Perrimon, N. (2009). Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila. Development 136(6): 983-93. PubMed Citation: 19211682

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

Dutriaux, A., Godart, A., Brachet, A. and Silber, J. (2013). The insulin receptor is required for the development of the Drosophila peripheral nervous system. PLoS One 8: e71857. PubMed ID: 24069139

Fernandez, R., et al. (1995). The Drosophila insulin receptor homolog: a gene essential for embryonic development encodes two receptor isoforms with different signaling potential. EMBO J. 14(14): 3373-3384. 7628438

Fernandez-Almonacid, R. and Rosen, O. M. (1987). Structure and ligand specificity of the Drosophila melanogaster insulin receptor. Molec. Cell. Biol. 7: 2718-2727. 3118188

Fernandez, A. M., et al. (2001). Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes. Genes Dev. 15: 1926-1934. 11485987

Gancz, D. and Gilboa, L. (2013). Insulin and Target of rapamycin signaling orchestrate the development of ovarian niche-stem cell units in Drosophila. Development 140: 4145-4154. PubMed ID: 24026119

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

Garcia-Caceres, C., et al. (2016). Astrocytic insulin signaling couples brain glucose uptake with nutrient availability. Cell 166: 867-880. PubMed ID: 27518562

Garofalo, R. S. and Rosen, O. M. (1988). Tissue localization of Drosophila melanogaster insulin receptor transcripts during development. Molec. Cell. Biol. 8: 1638-1647. 2454394

Gorczyca, M., Augart, C. and Budnik, V. (1993). Insulin-like receptor and insulin-like peptide are localized at neuromuscular junctions in Drosophila. J. Neurosci. 13(9): 3692-3704. 8366341

Gronke S., et al. Partridge L. (2010). Molecular evolution and functional characterization of Drosophila insulin-like peptides. PLoS Genet. 6: e1000857. PubMed Citation: 20195512

Gu, T., Zhao, T. and Hewes, R. S. (2014). Insulin signaling regulates neurite growth during metamorphic neuronal remodeling. Biol Open 3: 81-93. PubMed ID: 24357229

Hetru, C., et al. (1991). Isolation and structural characterization of an insulin-related molecule, a predominant neuropeptide from Locusta migratoria Eur. J. Biochem. 201: 495-499

Hirabayashi, S., Baranski, T. J. and Cagan, R. L. (2013). Transformed Drosophila cells evade diet-mediated insulin resistance through wingless signaling. Cell 154: 664-675. PubMed ID: 23911328

Hirabayashi, S. and Cagan, R. L. (2015). Salt-inducible kinases mediate nutrient-sensing to link dietary sugar and tumorigenesis in Drosophila. Elife 4. PubMed ID: 26573956

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., LaFever, L. and Drummond-Barbosa, D. (2008). Diet controls normal and tumorous germline stem cells via insulin-dependent and -independent mechanisms in Drosophila. Dev. Biol. 313: 700-712. PubMed Citation: 18068153

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(4): 1117-21. 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

Hung, W. L., Wang, Y., Chitturi, J. and Zhen, M. (2014). A Caenorhabditis elegans developmental decision requires insulin signaling-mediated neuron-intestine communication. Development 141: 1767-1779. PubMed ID: 24671950

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

Ikeya, T., et al. (2002). Nutrient-dependent expression of Insulin-like Peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila. Curr. Biol. 12: 1293-1300. 12176357

Kawakami, A., et al. (1989). Structure and organization of four clustered genes that encode bombyxin, an insulin-related brain secretory peptide of the silkmoth Bombyx mori Proc. Natl. Acad. Sci. 86: 6843-6847. 2674935

Kim, H., Rogers, M. J., Richmond, J. E. and McIntire, S. L. (2004). SNF-6 is an acetylcholine transporter interacting with the dystrophin complex in Caenorhabditis elegans. Nature 430: 891-896. Medline abstract: 15318222

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

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

Lebreton, S., Carlsson, M. A. and Witzgall, P. (2017). Insulin signaling in the peripheral and central nervous system regulates female sexual receptivity during starvation in Drosophila. Front Physiol 8: 685. PubMed ID: 28943854

Lee, R. Y., Hench, J. and Ruvkun, G. (2001). Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr. Biol. 11(24):1950-7. 11747821

Lee, S. S., Kennedy, S., Tolonen, A. C. and Ruvkun, G. (2003). DAF-16 target genes that control C. elegans life-span and metabolism. Science 300(5619): 644-7. 12690206

LaFever. L. and Drummond-Barbosa, D. (2005). Direct control of germline stem cell division and cyst growth by neural insulin in Drosophila. Science 309: 1071-1073. Medline abstract: 16099985

Leibiger, B., et al. (2001). Selective insulin signaling through a and b insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic cells. Molec. Cell 7: 559-570. 11463381

Leinwand, S. G. and Chalasani, S. H. (2013). Neuropeptide signaling remodels chemosensory circuit composition in Caenorhabditis elegans. Nat Neurosci 16: 1461-1467. PubMed ID: 24013594

Lin, K., Dorman, J.B., Rodan, A., and Kenyon, C. (1997). daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278: 1319-1322. 9360933

Lin, K., Hsin, H., Libina, N. and Kenyon, C. (2001). Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nat. Genet. 28(2): 139-45. 11381260

Lopez, A. L., Chen, J., Joo, H. J., Drake, M., Shidate, M., Kseib, C. and Arur, S. (2013). DAF-2 and ERK Couple Nutrient Availability to Meiotic Progression during Caenorhabditis elegans Oogenesis. Dev Cell 27: 227-240. PubMed ID: 24120884

Luo, J., Liu, Y. and Nassel, D. R. (2013). Insulin/IGF-Regulated Size Scaling of Neuroendocrine Cells Expressing the bHLH Transcription Factor Dimmed in Drosophila. PLoS Genet 9: e1004052. PubMed ID: 24385933

Mann K., et al. (2012). A putative tyrosine phosphorylation site of the cell surface receptor Golden goal is involved in synaptic layer selection in the visual system. Development Feb;139(4): 760-71. PubMed Citation: 22241840

Marin-Hincapie, M. and Garofalo, R. S. (1995). Drosophila insulin receptor: Lectin-binding properties and a role for oxidation-reduction of receptor thiols in activation. Endocrinology 136(6): 2357-2366. 7750456

Marin-Hincapie, M. and Garofalo, R. S. (1999). The carboxyl terminal extension of the Drosophila insulin receptor homologue binds IRS-1 and influences cell survival. J. Biol. Chem. 274(35): 24987-24994. 10455177

Marr, M. T., D'Alessio, J. A., Puig, O. and Tjian, R. (2007). IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev. 21(2): 175-83. Medline abstract: 17234883

Masumura, M., Ishizaki, H., Nagata, K., Kataoka, H., Suzuki, A. and Mizoguchi, A. (1997). Glucose stimulates the release of bombyxin, an insulin-related peptide of the silkworm Bombyx mori. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 118: 349-357. 9440228

Masumura, M., et al. (2000). Glucose stimulates the release of bombyxin, an insulin-related peptide of the silkworm Bombyx mori. Gen. Comp. Endocrinol. 118: 393-399. 10843790

McNeill H., Craig G. M. and Bateman J. M. (2008). Regulation of neurogenesis and epidermal growth factor receptor signalling by the insulin receptor/target of rapamycin pathway in Drosophila. Genetics 179: 843-853. PubMed Citation: 18505882

Montanaro, F. and Carbonetto, S. (2003). Targeting dystroglycan in the brain. Neuron 37: 193-196. Medline abstract: 12546815

Murakami, S. and Johnson, T. E. (2001). The OLD-1 positive regulator of longevity and stress resistance is under DAF-16 regulation in Caenorhabditis elegans. Curr. Biol. 11(19): 1517-23. 11591319

Murillo-Maldonado, J. M., Zeineddine, F. B., Stock, R., Thackeray, J. and Riesgo-Escovar, J. R. (2011). Insulin receptor-mediated signaling via phospholipase C-γ regulates growth and differentiation in Drosophila. PLoS One. 6(11): e28067. PubMed Citation: 22132213

Neumuller, R. A., Betschinger, J., Fischer, A., Bushati, N., Poernbacher, I., Mechtler, K., Cohen, S. M. and Knoblich, J. A. (2008). Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage. Nature 454: 241-245. PubMed Citation: 18528333

Nishida, Y., et al. (1986). Cloning of a Drosophila cDNA encoding a polypeptide similar to the human insulin receptor precursor. Biochem. biophys. Res. Commun. 141: 474-481. 3099787

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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

Oldham, S., et al. (2002). The Drosophila insulin/IGF receptor controls growth and size by modulating PtdInsP3 levels. Development 129: 4103-4109. 12163412

Ookuma, S., Fukuda, M. and Nishida, E. (2003). Identification of a DAF-16 transcriptional target gene, scl-1, that regulates longevity and stress resistance in Caenorhabditis elegans. Curr. Biol. 13(5): 427-31. 1262019

Park, S., Alfa, R. W., Topper, S. M., Kim, G. E., Kockel, L. and Kim, S. K. (2014). A genetic strategy to measure circulating Drosophila insulin reveals genes regulating insulin production and secretion. PLoS Genet 10: e1004555. PubMed ID: 25101872

Piovant, M., and Lena, P. (1988). Membrane glycoproteins immunologically related to the human insulin receptor are associated with presumptive neuronal territories and developing neurones in Drosophila melanogaster. Development. 103(1): 145-56. 3143540

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Petruzzelli, L., et al. (1986). Isolation of a Drosophila genomic sequence homologous to the kinase domain of the human insulin receptor and detection of the phosphorylated Drosophila receptor with an anti-peptide antibody. Proc. Natl. Acad. Sci. 83(13): 4710-4. 3014506

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

Pimentel, B., et al. (1996). Insulin acts as an embryonic growth factor for Drosophila neural cells. Biochem. biophys. Res. Commun. 226(3): 855-861. 8831701

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Puig, O., Marr, M. T., Ruhf, R. L. and Tjian, R. (2003). Control of cell number by Drosophila FOXO: downstream and feedback regulation of the insulin receptor pathway. Genes and Development 17: 2006-2020. 12893776

Puig, O. and Tjian, R. (2005). Transcriptional feedback control of insulin receptor by dFOXO/FOXO1. Genes Dev. 19(20): 2435-46. 16230533

Qu, Q. and Smith, F. I. (2004). Alpha-dystroglycan interactions affect cerebellar granule neuron migration. J. Neurosci. Res. 76: 771-782. Medline abstract: 15160389

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

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Rauschenbach, I. Y., Karpova, E. K., Adonyeva, N. V., Andreenkova, O. V., Faddeeva, N. V., Burdina, E. K., Alekseev, A. A., Menshanov, P. N. and Gruntenko, N. E. (2014). Disruption of insulin signalling affects the neuroendocrine stress reaction in Drosophila females. J Exp Biol 217(Pt 20):3733-41. PubMed ID: 25214494

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Ryuda, M., et al. (2011). Identification of a novel gene, anorexia, regulating feeding activity via insulin signaling in Drosophila melanogaster. J. Biol. Chem. 286(44): 38417-26. PubMed Citation: 21917925

Samuelson, A. V., Carr, C. E. and Ruvkun, G. (2007). Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants. Genes Dev. 21(22): 2976-94. PubMed citation: 18006689

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Shakhmantsir, I., Massad, N. L. and Kennell, J. A. (2013). Regulation of cuticle pigmentation in Drosophila by the nutrient sensing insulin and TOR signaling pathways. Dev Dyn. [Epub ahead of print] PubMed ID: 24133012

Shcherbata, H. R., et al. (2007). Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy. EMBO J. 26(2): 481-93. Medline abstract: 17215867

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Sim, C. and Denlinger, D. L. (2008). Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens. Proc. Natl. Acad. Sci. 105(18): 6777-81. PubMed Citation: 18448677

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

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Wehr, M. C., Holder, M. V., Gailite, I., Saunders, R. E., Maile, T. M., Ciirdaeva, E., Instrell, R., Jiang, M., Howell, M., Rossner, M. J. and Tapon, N. (2013). Salt-inducible kinases regulate growth through the Hippo signalling pathway in Drosophila. Nat Cell Biol 15: 61-71. PubMed ID: 23263283

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Yu, H. and Larsen, P. L. (2001). DAF-16-dependent and independent expression targets of DAF-2 insulin receptor-like pathway in Caenorhabditis elegans include FKBPs. J. Mol. Biol. 314(5): 1017-28. 11743719

Yu, J. Y., et al. (2009). Dicer-1-dependent Dacapo suppression acts downstream of Insulin receptor in regulating cell division of Drosophila germline stem cells. Development 136(9): 1497-507. PubMed Citation: 19336466

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

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

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