Insulin-like peptide 6: Biological Overview | References
Gene name - Insulin-like peptide 6
Synonyms - DILP6
Cytological map position - 3A1-3A1
Function - ligand
Symbol - Ilp6
FlyBase ID: FBgn0044047
Genetic map position - X: 2,225,439..2,227,719 [-]
Classification - Insulin like peptide
Cellular location - secreted
|Recent literature||Okamoto, N. and Nishimura, T. (2015). Signaling from glia and cholinergic neurons controls nutrient-dependent production of an insulin-like peptide for Drosophila body growth. Dev Cell 35: 295-310. PubMed ID: 26555050
The insulin-like peptide (ILP) family plays key biological roles in the control of body growth. Although the functions of ILPs are well understood, the mechanisms by which organisms sense their nutrient status and thereby control ILP production remain largely unknown. This study shows that signaling relay and feedback mechanisms control the nutrient-dependent expression of Drosophila ILP5 (Dilp5). The expression of dilp5 in brain insulin-producing cells (IPCs) is negatively regulated by the transcription factor FoxO. Glia-derived Dilp6 remotely regulates the FoxO activity in IPCs, primarily through Jeb secreted by cholinergic neurons. Dilp6 production by surface glia is amplified by cellular response to circulating Dilps derived from IPCs, in concert with amino acid signals. The induction of dilp5 is critical for sustaining body growth under restricted food conditions. These results provide a molecular framework that explains how the production of an endocrine hormone in a specific tissue is coordinated with environmental conditions.
|Aradhya, R., Zmojdzian, M., Da Ponte, J. P. and Jagla, K. (2015). Muscle niche-driven Insulin-Notch-Myc cascade reactivates dormant adult muscle precursors in Drosophila. Elife 4 [Epub ahead of print]. PubMed ID: 26650355
How stem cells specified during development keep their non-differentiated quiescent state, and how they are reactivated, remain poorly understood. This study applied a Drosophila model to follow in vivo behavior of Adult Muscle Precursors (AMPs), the transient fruit fly muscle stem cells. Emerging AMPs send out thin filopodia that make contact with neighboring muscles. AMPs keep their filopodia-based association with muscles throughout their dormant state but also when they start to proliferate, suggesting that muscles could play a role in AMP reactivation. Indeed, genetic analyses indicate that muscles send inductive dIlp6 signals that switch the Insulin pathway ON in closely associated AMPs. This leads to the activation of Notch, which regulates AMP proliferation via dMyc. Altogether, this study reports that Drosophila AMPs display homing behavior to muscle niche and that the niche-driven Insulin-Notch-dMyc cascade plays a key role in setting the activated state of AMPs.
|Rauschenbach, I. Y., Karpova, E. K., Burdina, E. V., Adonyeva, N. V., Bykov, R. A., Ilinsky, Y. Y., Menshanov, P. N. and Gruntenko, N. E. (2016). Insulin-like peptide DILP6 regulates juvenile hormone and dopamine metabolism in Drosophila females. Gen Comp Endocrinol 243: 1-9. PubMed ID: 27823956
Insulin-like peptide DILP6 is a component of the insulin/insulin-like growth factor signalling pathway of Drosophila. Juvenile hormone (JH) and dopamine (DA) are involved in the stress response and in the control of reproduction. This study investigated whether DILP6 regulates the JH and DA levels by studying the effect of a strong hypomorphic mutation dilp641 on JH and DA metabolism in D. melanogaster females. DILP6 was shown to regulate JH and DA metabolism: the mutation dilp641 results in a reduction in JH-hydrolysing activity and an increase in the activities of DA synthesis enzymes (alkaline phosphatase (ALP) and tyrosine hydroxylase (TH)). In the mutant females, increased fecundity was found, in addition to the intensity of the response (stress reactivity) of ALP and TH to heat stress. This suggests an increased level of JH synthesis. This suggestion was confirmed by treating the mutant females with the JH inhibitor, precocene, which restores the activity and stress reactivity of ALP and TH as well as fecundity to levels similar to those in the control flies. The data suggest a feedback system in the interaction between JH and DILP6 in which DILP6 negatively regulates the JH titre via an increase in the hormone degradation and a decrease in its synthesis.
|Umezaki, Y., Hayley, S. E., Chu, M. L., Seo, H. W., Shah, P. and Hamada, F. N. (2018). Feeding-state-dependent modulation of temperature preference requires insulin signaling in Drosophila warm-sensing neurons. Curr Biol 28(5): 779-787.e773. PubMed ID: 29478858
Starvation is life-threatening and therefore strongly modulates many aspects of animal behavior and physiology. In mammals, hunger causes a reduction in body temperature and metabolism, resulting in conservation of energy for survival. However, the molecular basis of the modulation of thermoregulation by starvation remains largely unclear. Whereas mammals control their body temperature internally, small ectotherms, such as Drosophila, set their body temperature by selecting an ideal environmental temperature through temperature preference behaviors. This study demonstrates in Drosophila that starvation results in a lower preferred temperature, which parallels the reduction in body temperature in mammals. The insulin/insulin-like growth factor (IGF) signaling (IIS) pathway is involved in starvation-induced behaviors and physiology and is well conserved in vertebrates and invertebrates. Insulin-like peptide 6 (Ilp6) in the fat body (fly liver and adipose tissues) is responsible for the starvation-induced reduction in preferred temperature (Tp). Temperature preference behavior is controlled by the anterior cells (ACs), which respond to warm temperatures via transient receptor potential A1 (TrpA1). This study demonstrated that starvation decreases the responding temperature of ACs via insulin signaling, resulting in a lower Tp than in nutrient-rich conditions. Thus, this study shows that hunger information is conveyed from fat tissues via Ilp6 and influences the sensitivity of warm-sensing neurons in the brain, resulting in a lower temperature set point. Because starvation commonly results in a lower body temperature in both flies and mammals, it is proposed that insulin signaling is an ancient mediator of starvation-induced thermoregulation.
|Doupe, D. P., Marshall, O. J., Dayton, H., Brand, A. H. and Perrimon, N. (2018). Drosophila intestinal stem and progenitor cells are major sources and regulators of homeostatic niche signals. Proc Natl Acad Sci U S A. PubMed ID: 30404917
Epithelial homeostasis requires the precise balance of epithelial stem/progenitor proliferation and differentiation. While many signaling pathways that regulate epithelial stem cells have been identified, it is probable that other regulators remain unidentified. This study uses gene-expression profiling by targeted DamID to identify the stem/progenitor-specific transcription and signaling factors in the Drosophila midgut. Many signaling pathway components, including ligands of most major pathways, exhibit stem/progenitor-specific expression and have regulatory regions bound by both intrinsic and extrinsic transcription factors. In addition to previously identified stem/progenitor-derived ligands, this study shows that both the insulin-like factor Ilp6 and TNF ligand eiger are specifically expressed in the stem/progenitors and regulate normal tissue homeostasis. It is proposed that intestinal stem cells not only integrate multiple signals but also contribute to and regulate the homeostatic signaling microenvironmental niche through the expression of autocrine and paracrine factors.
In metazoans, tissue growth relies on the availability of nutrients - stored internally or obtained from the environment - and the resulting activation of insulin/IGF signaling (IIS). In Drosophila, growth is mediated by seven Drosophila insulin-like peptides (Dilps), acting through a canonical IIS pathway. During the larval period, animals feed and Dilps produced by the brain couple nutrient uptake with systemic growth. This study shows that during metamorphosis, when feeding stops, a specific DILP (Dilp6) is produced by the fat body and relays the growth signal. Expression of DILP6 during pupal development is controlled by the steroid hormone ecdysone. Remarkably, DILP6 expression is also induced upon starvation, and both its developmental and environmental expression require the Drosophila FoxO transcription factor. This study reveals a specific class of ILPs induced upon metabolic stress that promotes growth in conditions of nutritional deprivation or following developmentally induced cessation of feeding (Slaidina, 2009).
Growth relies on the ability of cells and organisms to access nutrients. Nutrients can be obtained from diverse sources, such as from the environment through feeding, or from internal stores as with early embryos that develop from large eggs. Accordingly, because alternate sources of nutrients are used during specific periods of development, organisms must be able to adapt their metabolic and growth programs to changes in the developmental or environmental energy context (Slaidina, 2009).
In complex animal species, growth is controlled by intermingled paracrine and endocrine regulatory processes, with organ and tissue growth governed by specific genetic programs that determine the target size and relative proportions of the species. The output of these genetic programs is further modified by environmental cues, including nutrition. Variations in nutritional input can influence growth and metabolism via insulin/IGF signaling (IIS). In particular, when nutrients are abundant, IIS is maximally active and growth is limited solely by the organ-intrinsic program; upon nutrient shortage, in contrast, IIS becomes limiting and restricts the growth and metabolic parameters accordingly (Slaidina, 2009).
In mammals, the IIS system is split into two complementary and interacting subsystems that govern growth, metabolism, reproduction, and longevity (Nakae, 2001; Saltiel, 2001). The first of these corresponds to circulating insulin levels, which control carbohydrate and fat metabolism, and the second is the GH/IGF-I axis, which regulates cell and tissue growth. Starvation lowers circulating IGF-I, in part through decreased transcription of the IGF-I gene in the liver; this suggests that one major way in which starvation can affect growth is by reducing levels of circulating growth factors (Slaidina, 2009).
The function of IIS in growth control is remarkably conserved in insects, and in particular in Drosophila, where seven Drosophila insulin-like peptides (DILPs) have been identified (Géminard, 2006). The various DILP genes are expressed in different larval and adult tissues, suggesting that they carry nonredundant functions (Brogiolo, 2001). In particular, DILP1, -2, -3, and -5 are expressed in specialized neurosecretory cells located in each brain hemisphere, called the insulin-producing cells (IPCs). Genetic ablation of these cells leads to severe larval growth deficits, hypertrehalosemia, and increased lifespan (Slaidina, 2009).
One major role for IIS in insects is to couple growth with the animal's energy status. Indeed, total nutrient deprivation downregulates DILP3 and DILP5 transcription in the IPCs, although DILP2 expression remains unchanged. Recent results indicate that variations in nutritional information are relayed by a nutrient sensor operating in the fat body, a larval organ that shares metabolic functions with the vertebrate white fat and liver. In particular, it has been shown that amino acid restriction triggers fat body-specific inhibition of the TOR complex1 (TORC1) (Colombani, 2003), a major cell-based nutrient-sensing pathway. Inhibition of TORC1 in the fat body systemically reduces larval growth in part by blocking Dilp secretion from the brain IPCs (Géminard, 2009). Therefore, in line with the decreased levels of circulating IGF-I in vertebrates, starvation affects Drosophila growth by severely reducing brain-specific DILP function (Slaidina, 2009).
Interestingly, previous work has shown that protein starvation causes the growth arrest of endoreplicative larval tissues (ERTs), while only slowing the growth and proliferation of cells in the larval brain and in imaginal discs. Similarly, generally reduced TOR signaling in the larva, which in many respects mimics the starvation state, strongly inhibits ERT growth, while generally sparing the imaginal tissues (ITs) that form the adult structures. This suggests a protection mechanism whereby, under adverse nutrition conditions, the fat body allows larval resources to be reallocated to high-priority tissues like the imaginal discs. Significantly, such a mechanism would require that some ILPs are produced during starvation and activate IIS in the tissues that continue to grow (Slaidina, 2009).
Feeding arrest is also a programmed event during development. At the end of the larval period, animals undergo a stereotyped behavior called the wandering stage, when they migrate away from the food and prepare for pupariation. This developmentally induced starvation precedes the long pupal feeding arrest. During pupal development, larval tissues undergo intense remodeling. This process involves a major reallocation of resources, as future adult tissues form from ITs in a process that uses either nutrient stores that had accumulated in fat cells during larval life, or energy obtained from the degradation of obsolete larval tissues. Since organisms do not feed during this stage, no global growth or weight gain is observed; nevertheless, because tissue remodeling involves cell growth and proliferation, growth-promoting pathways presumably come into play. The paradox of pursuing a growth program in a nonfeeding organism that is subjected to catabolic regulation could be circumvented by the induction of growth-promoting hormones upon feeding arrest (Slaidina, 2009).
This study presents the characterization of a particular DILP, DILP6, which promotes growth during nonfeeding stages. The DILP6 gene is expressed in fat body cells and is strongly induced during the wandering larval and pupal periods, as well as upon starvation. Reduced DILP6 function results in a growth deficit during pupal development and an increased sensitivity to starvation in young adults. The sudden increase of DILP6 expression at the onset of pupal development requires an endocrine signal that is provided by the steroid hormone ecdysone. In parallel, starvation increases DILP6 expression through dFoxO-mediated feedback regulation of IIS. Therefore, DILP6 constitutes an IGF-like peptide with a specialized role in promoting growth during developmentally or environmentally induced nonfeeding states (Slaidina, 2009).
During the successive stages of development, organisms use alternate sources of nutrients to support tissue growth and morphogenesis. In Drosophila, embryonic tissues develop using maternal stores accumulated in the egg in the form of yolk. Larval development follows, with a major growth program relying on the animals' capacity to obtain nutrients from the environment. Finally, during the pupal stage, animals do not feed, and a large quantity of nutrients stored in fat cells allows pupae to prolong growth and finalize the development of adult structures. On top of these basic developmental strategies, feeding larvae have evolved additional buffering mechanisms to protect growing tissues from sudden variations in environmental energy supplies. Notably, brain ILPs promote larval growth and allow the coupling of growth to nutritional input. Their expression and secretion from brain IPCs decrease upon starvation (Géminard, 2009), and several brain DILPs show only residual expression in the pupa. Therefore, there must be a distinct set of growth inducers that take the lead to activate growth in the pupa and upon nutritional stress. In both of these contexts, a physiological switch takes place that triggers the activation of DILP6, a member of a distinct class of ILPs devoted to growth during nonfeeding periods (Slaidina, 2009).
The DILP1 and DILP3 genes are also expressed during pupal development, suggesting that they may act in concert with DILP6. Individual knockout of either of these two DILP genes produces only marginal growth defects, suggesting that there is a high level of redundancy between them or with DILP6. The observation that DILP1 expression increases two-fold in DILP6 mutant larvae suggests a possible compensatory mechanism that could partially suppress the growth impairment observed in DILP6 mutants. The functional class of ILPs represented by DILP6 may be conserved in other insect species, as an ecdysone-induced, fat-body-specific ILP has recently been described (Okamoto, 2009) in Bombyx mori (Slaidina, 2009).
The developmental and environmental induction of DILP6 involves overlapping mechanisms. First, in response to nutrient deprivation, the IIS component, dFoxO, provokes a burst of DILP6 transcription, thereby linking DILP6 expression with the nutritional status of the animal. This represents a feedback regulation on IIS, as dFoxO, an inhibitor in the IIS pathway, induces the expression of DILP6, an activator of IIS. Interestingly, expression of DILP3 in the adult was also recently shown to depend on dFoxO function, suggesting that other DILP genes in this subclass are subjected to similar controls (Slaidina, 2009).
DILP6 does not appear to be effective as a paracrine/autocrine factor for fat cells. Indeed, fat cells of starved larvae, which express high levels of DILP6, undergo extensive autophagic transformation, even though autophagy has been shown to be blocked in these cells by IIS activation (Rusten, 2004; Scott, 2004). In addition, overexpression of DILP6 in the fat body of starved larvae does not prevent autophagy (Slaidina, 2009).
More generally, ERTs present stronger growth inhibition in response to starvation than do ITs. The role of a starvation-specific ILP that is induced upon nutritional stress could be to reroute energy stores toward high-priority organs and tissues, such as those responsible for the formation of the future adult. The specific action of DILP6 on imaginal cells could contribute to this diversified behavior, although this would require that ITs are more receptive to the DILP6 signal than are ERTs, at least upon starvation. Such differences in the response of ERTs and ITs to the DILP signal, combined with the production of specific DILPs upon starvation, could constitute a bona fide mechanism for the specific allocation of spare resources to ITs under nutritional stress. However, the mechanisms for such a biased response need to be elucidated (Slaidina, 2009).
At the end of larval development, animals stop feeding and prepare for pupal development. This study shows that tissue remodeling in the pupa involves IIS-dependent growth, and that DILP6 is specifically expressed and required for growth during this period. The transition from larval to pupal development is controlled by the steroid hormone ecdysone (20E), and it was also shown that 20E is required for proper DILP6 induction at the larval/pupal transition. In view of the absence of obvious EcR/Usp binding sites in the 5' region of the DILP6 gene, as well as a previous demonstration that EcR signaling controls dFoxO nuclear localization (Colombani, 2005), it is hypothesized that dFoxO could mediate the ecdysone-dependent expression of DILP6. However, both genetics and ex vivo experiments on dissected fat bodies indicate that, although dFoxO appears to contribute to the developmental induction of DILP6 at the larval/pupal transition, it is not required for the 20E-induced expression of DILP6. In an accompanying manuscript, Okamoto (2009) reports that 20E-induced expression of DILP6 is not affected by cycloheximide, suggesting that the transcriptional induction of DILP6 by EcR/Usp is direct (Slaidina, 2009).
It has been previously shown that ecdysone has a growth-inhibitory function during larval development (Colombani, 2005). Indeed, increased basal levels of circulating ecdysone in larvae can reduce the growth rate and, conversely, decreased basal ecdysone levels can increase the growth rate. Although the mechanisms underlying this relationship are not yet fully understood, this study has established that the levels of ecdysone produced experimentally in these experiments remain close to basal levels, and are insufficient to modify DILP6 expression. Therefore, while basal levels of ecdysone can inhibit systemic growth through an unknown mechanism, high ecdysone levels at the larval/pupal transition can induce DILP6, and thus systemically activate IIS (Slaidina, 2009).
One puzzling observation reported in this study is that the modification of DILP6 expression in pupae can alter the adult mass as well as the resistance of animals to starvation at eclosion. How can DILP6 overexpression in pupae increase adult mass if the mass of the pupa is fixed at the end of larval life? One possible explanation is that DILP6 participates in a tradeoff between the construction of adult tissues and the maintenance of energy stores in the pupa. Indeed, the levels of both TAG and glycogen stores in the young adult are affected by DILP6 levels in the pupa. In this line, recent reports indicate that, under optimal conditions, not all nutrients are used by the pupa, and part of the energy is conserved to provide sustenance during the early period of adult life that precedes feeding. Some larval fat body cells are still present in early adults, and provide energy until feeding begins. Suppressing the death of these cells increases the energy stores and enhances the resistance of young adults to starvation (Aguila, 2007). DILP6 knockdown in pupae has a similar effect: less energy is used by the pupa to build tissues, meaning that the adult ecloses with a smaller body, but with greater energy stores to help overcome early nutritional stress. DILP6 overexpression has the opposite effect. The current results therefore indicate that DILP6 sets the energy balance in pupae by promoting tissue growth, while sparing an energy pool that can be used by the young adult (Slaidina, 2009).
DILP6 shares some specific features with vertebrate IGF-I that distinguish both of them from insulin. DILP6 peptide sequence does not present obvious cleavage sites for an internal C peptide (Brogiolo, 2001). It is produced in the fat body, a tissue sharing common functions with the vertebrate liver, where IGF-I is mainly produced. DILP6 mutant animals present growth defects without obvious metabolic changes, suggesting that DILP6 might have an exclusive growth function. Finally, the induction of growth factor production under conditions of energy stress is also relevant to cancer biology. Indeed, IGF-I and IGF-II are frequently expressed within neoplastic tissue. It is suspected that they act as autocrine and paracrine growth factors within tumors, allowing tumor cells to evade nutritional shortage and acquire survival properties (Pollak, 2008). The induction of DILP6 under starvation and its preferential targeting to ITs instead of ERTs could represent an interesting parallel to the induction of IGFs in tumor cells, where the selective action of growth factors can promote growth and survival of specific tissues in a nonfavorable environment (Slaidina, 2009).
Members of the insulin family of peptides have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. This study sshow that Drosophila insulin-like peptide 6 (DILP6), which is structurally similar to vertebrate insulin-like growth factor (IGF), is predominantly expressed in the fat body, a functional equivalent of the vertebrate liver and adipocytes. This expression occurs during the postfeeding stage under the direct regulation of ecdysteroid. dilp6 mutants show growth defects during the postfeeding stage, that result in reduced adult body size through a decrease in cell number. This phenotype is rescued by fat body-specific expression of dilp6. These data indicate that DILP6 is a functional, as well as a structural, counterpart of vertebrate IGFs. The data provide in vivo evidence for a role of ILPs in determining adult body size through the regulation of postfeeding growth (Okamoto, 2009b).
DILP6, one of seven ILPs in Drosophila, is produced primarily in the fat body to regulate postfeeding growth without affecting the timing of metamorphosis. This observation is interesting to consider in light of previous findings that suggest that insulin/IGF signaling (IIS) affects both the timing of metamorphosis and the rate of growth. These results thus clearly demonstrate that different ILPs have distinct temporal roles during development. Similar results are also presented in a second publication (Slaidina, 2009; Okamoto, 2009b and references therein).
Insects utilize larval accumulated nutrients for the development of adult-specific tissues during the postfeeding period. How DILP6 mediates this tissue-specific growth remains unknown, but previous reports indicate interplays between 20E and IIS involved in this process. In the fat body, 20E antagonizes IIS, which probably blocks an autocrine effect of DILP6. In contrast, 20E synergistically enhances IIS in the imaginal disks to promote growth. It is also interesting to note that the downregulation of IIS by 20E in the fat body activates autophagy, which promotes the release of stored nutrients. It is suggested that these tissue-specific effects of 20E on IIS facilitate the directional transfer of nutrients from storage organs (fat body) to developing disks to promote adult-specific tissue growth. DILP6 appears to play a pivotal role in this process, and its loss leads to enhanced excretion of unused materials during wandering and after eclosion (Okamoto, 2009b).
The independent role of DILP6 compared to IPC-derived DILPs is reminiscent of the roles of IGFs compared to insulin in mammals. There are three major aspects of their similarities. First, it was shown that dilp6 is predominantly expressed in the fat body, a functional equivalent of the mammalian liver and adipose tissue, and the liver is the principal source of circulating IGFs in mammals. Second, the data revealed that the expression of dilp6 is directly regulated by the steroid hormone, 20E, when growth is independent of extrinsic nutritional input. Although the expression of IGFs can be regulated by nutrition, high concentrations of IGF-I and -II are observed during pubertal and fetal development, respectively, reflecting their importance in these key developmental transitions in mammals. Moreover, igf-I expression in several organs is induced by sex steroids, further supporting the analogy between DILP6 and IGFs. Third, the predicted peptide structure of DILP6 is distinct from other DILPs in that it has a short C peptide, which is more similar to vertebrate IGFs than to insulin. Moreover, the short C peptide is likely to remain in the mature form like IGFs, because of the lack of a cleavage site. Thus, the structural aspect also favors the analogy between DILP6 and IGFs. From all these similarities between DILP6 and IGFs, it is proposed that DILP6 is a functional as well as a structural counterpart of vertebrate IGFs, and therefore DILP6 is defined as a Drosophila IGFLP. It should be noted here that, in parallel with the analogy between DILP6 and IGFs, there are several analogies between IPC-derived DILPs and insulin in terms of the source tissues and the nutritional regulation of the expression and peptide secretion (Okamoto, 2009b and references therein).
Together with a previous characterization of IGFLP in Bombyx (Okamoto, 2009a), it is highly likely that IGFLP is widely present in divergent insect orders. Surprisingly, however, phylogenetic analysis supports no orthology between BIGFLP and DILP6, suggesting that BIGFLP and DILP6 have evolved independently. It is hypothesized that, in ancestral insect species, there was a single ILP that was expressed both in the brain IPCs and in the fat body. This ancestral ILP was probably under distinct regulatory mechanisms (nutritional and developmental) in these tissues, which facilitated functional diversification of IPC-derived ILPs and fat body-derived ILPs after a gene duplication event(s) that happened independently in each insect order. In the previous study in Bombyx, it was demonstrated that BIGFLP is released as a single-chain polypeptide, despite having two potential cleavage sites within the C domain (Okamoto, 2009a). This suggests the lack of processing enzymes to generate mature insulin in the fat body, which probably explains why fat body-derived ILPs in different species have attained similar structural features as IGFLPs (shortened C-peptide and/or the loss of cleavage sites) despite their independent lineages. Studies in orthopteran species (which are considered closer to earlier insect species), where there is only one identified ILP the expression of which is differentially regulated in the brain IPCs and in the fat body, support the hypothesis (Okamoto, 2009b).
Since most insect genomes contain a single insulin/IGF-like receptor gene, IGFLPs and the other ILPs presumably activate the same receptor, although its binding affinities for different ligands likely vary according to the distinct structural features of the ligands. In contrast, mammalian genomes contain multiple receptors, each of which responds to one primary ligand. Therefore, there also appears to exist a clear difference between mammalian IGFs and insect IGFLPs. Considering the pivotal role of IGFs/IGFLPs during development in both of these animal groups, further investigations of the similarities as well as the differences in these signaling pathways should enrich the understanding of underlying mechanisms that control development throughout the animal kingdom (Okamoto, 2009b).
Insulin/Insulin-like growth factor signaling regulates homeostasis and growth in mammals, and is implicated in diseases from diabetes to cancer. In Drosophila, as in other invertebrates, multiple Insulin-Like Peptides (DILPs) are encoded by a family of related genes. To assess DILPs' physiological roles, small deficiencies were generated that uncover single or multiple dilps, generating genetic loss-of-function mutations. Deletion of dilps1-5 generated homozygotes that are small, severely growth-delayed, and poorly viable and fertile. These animals display reduced metabolic activity, decreased triglyceride levels and prematurely activate autophagy, indicative of 'starvation in the midst of plenty,' a hallmark of Type I diabetes. Furthermore, circulating sugar levels are elevated in Df [dilp1-5] homozygotes during eating and fasting. In contrast, Df[dilp6] or Df[dilp7] animals showed no major metabolic defects. Physiological differences between mammals and insects may explain the unexpected survival of lean, 'diabetic' flies (Zhang, 2009. Full text of article).
Insulin/IGF signaling (IIS) is highly conserved throughout the animal kingdom and is important for regulation of growth and metabolism in a range of organisms. In most cases, IIS receptors, IIS ligands, or both are represented by multigene families. In mice, where this has been examined in most detail, family members have both distinct and overlapping roles in regulating animal physiology. Insulin-like activities were identified in invertebrates, including Drosophila, many years ago. Based upn genomic sequence and similarity to mammalian insulin, Drosophila have at least seven candidate IIS ligands. Evidence from RNAi experiments indicates a high degree of redundancy among DILPs, including compensatory upregulation of expression of dilp gene(s), when others are experimentally downregulated. Thus, multiple approaches will be required to assess the functions of this complex multigene family. As a step toward defining DILP wild-type functions, this study has taken a loss-of-function genetic approach that makes use of FRT sites in the Drosophila genome to generate small deficiencies that uncover genes of interest, followed by 'adding back' a dilp gene to test for functional rescue. Many of the effects found to be associated with loss-of-DILP-function are reminiscent of defects in mammalian IIS. Df[dilp1-5] homozygotes exhibit growth defects, with decreases in overall body size due to decreases in cell size and cell number; developmental delay; and poor fertility and viability. In keeping with DILPs functioning as DInR ligands, tissue-specific overexpression of DInR promotes growth and some combinations of dinr alleles support survival but transheterozygotes are small and growth delayed, similar to Df[dilp1-5] animals and chico mutants. Similar growth defects are observed in IGF-1 and IGF-1R null mice, which are developmentally delayed but viable. Interestingly, mouse IR and Ins null mutants are also small at birth, although larger than IGF-1/IGF-1R mutants. Similarly, several human syndromes point to a role for IR in growth regulation in humans: for example, Leprechaunism is associated with severe growth retardation and results from mutations in IR (Zhang, 2009).
Metabolic control by IIS also shows many similarities between the Drosophila and mammalian systems. In mice, knock-out of the insulin receptor or the insulin genes resulted in diabetes, accompanied by hyperglycemia and ketoacidosis, resulting in perinatal lethality. These features phenocopy human Type I diabetes in which insulin production gradually fails. The Df[dilp1-5] homozygotes examined in this stuyd show many defects similar to those in mammals: leanness, inappropriate breakdown of fat tissue, and high levels of circulating sugar. These results demonstrate parallels in metabolic regulation between Drosophila and mammals. Furthermore, the counterregulatory hormone to insulin, glucagon, appears to be functionally conserved in insects. Insect adipokinetic hormone (AKH) appears to act like mammalian glucagon by stimulating gluconeogenesis, as ablation of the corpora cardiaca cells that produce AKH caused decreased levels of sugar without affecting growth or developmental time (Zhang, 2009).
Unlike mammals with defective insulin or insulin genes, Df[dilp1-5] homozygotes survive and are fertile. At this point, the possibility cannot be ruled out that compensatory action of DILP6 and/or DILP7 explains the survival of Df[dilp1-5] homozygotes, although neither dilp6 nor dilp7 mutant larvae showed metabolic defects. However, irrespective of this, it is clear that Df[dilp1-5] homozygotes do indeed display diabetic-like abnormalities, and it is thus surprising that they are not more severely affected. For example, even though these animals are breaking down fat, as evidenced by lower whole body triglycerides and activation of fat body autophagy, they do not appear to be suffering from acute effects of ketoacidosis that are toxic in mammals. Similarly, these animals appear relatively resistant to negative impacts of persistent hyperglycemia. This differential tolerance to long-term and endemic diabetic manifestations may reflect physiological differences between mammals and insects. First, the primary circulating sugar in insects is trehalose, a nonreducing disaccharide that will not generate glycation products, which are the cause of many of the long-term complications seen in human diabetes. Second, insects have endogenous mechanisms to raise sugar levels, and, in contrast to the situation for mammals where this increase is highly deleterious, this actually promotes the survival of insects under harsh conditions. For example, one of the mechanisms used by many insects to tolerate cold temperatures is the induction of high levels of polyols such as glycerol, or sugars, including trehalose, sorbitol, and others, that act as cryoprotectants. The levels of these polyols and sugars vary over seasons, with levels increasing in the autumn as temperatures get colder. Levels then decline in the spring, once the animal has survived cold conditions over winter. In keeping with this, a recent study showed that injection of trehalose enhanced resistance to heat and cold stress and dehydration in the Antarctic midge. This physiological rise in levels of these cryoprotectants in winter is associated with induction of diapause, another mechanism specific to invertebrates that allows animals to survive harsh conditions. It is of interest in this context to note that inhibition of IIS pathways induced diapause in mosquitoes. Future studies will be necessary to determine the long-term effects of elevated circulating sugar levels on fly physiology and to determine whether conserved molecular and biochemical pathways, shared by insects and mammals, account for the abnormalities in sugar and fat homeostasis seen in dilp mutants (Zhang, 2009).
The systemic regulation of stem cells ensures that they meet the needs of the organism during growth and in response to injury. A key point of regulation is the decision between quiescence and proliferation. During development, Drosophila neural stem cells (neuroblasts) transit through a period of quiescence separating distinct embryonic and postembryonic phases of proliferation. It is known that neuroblasts exit quiescence via a hitherto unknown pathway in response to a nutrition-dependent signal from the fat body. This study has identified a population of glial cells that produce insulin/IGF-like peptides in response to nutrition, and shows that the insulin/IGF receptor pathway is necessary for neuroblasts to exit quiescence. The forced expression of insulin/IGF-like peptides in glia, or activation of PI3K/Akt signaling in neuroblasts, can drive neuroblast growth and proliferation in the absence of dietary protein and thus uncouple neuroblasts from systemic control (Chell, 2010).
A transcriptome analysis comparing VNCs from newly hatched larvae and VNCs from larvae at the end of the first instar suggested that the expression of dILP6 and dILP2 increases in the VNC during neuroblast reactivation. The seven dILPs are expressed in distinct spatiotemporal patterns during development. dILP6 is reported to be expressed in the larval gut and the pupal fat body , whereas dILP2 is known to be expressed in the IPC neurons of the brain (along with dilps 1, 3, and 5). To determine whether dILP6 is also expressed in the CNS, a dilp6-GAL4 line was generated. dilp6-GAL4 drives expression in a subset of the surface glia that wraps the CNS. Strong expression was evident by mid first instar and was maintained throughout neuroblast reactivation. The expression of dILP2 was assayed by immunohistochemistry; it too was expressed in the same surface glial population. The glial cells labeled by dilp6-GAL4 are located above the neuroblasts and underneath the surrounding basement membrane. They are stellate in appearance, with several processes radiating from the central cell body. Thus, dILPs, expressed by glial cells, are ideally positioned to activate the dInR pathway in neuroblasts during reactivation (Chell, 2010).
Drosophila neuroblasts in the central brain and thoracic ventral nerve cord (tVNC) are quiescent for 24 hours between their embryonic and larval phases of proliferation. Quiescent neuroblasts are easily identifiable and are amenable to genetic manipulation, making them a potentially powerful model with which to study the transition between quiescence and proliferation. However, the mechanisms regulating the exit from quiescence, either intrinsic or extrinsic, are not well established. Genetic studies found that Drosophila FGF, in concert with Drosophila Perlecan, promotes the neuroblast transition from quiescence to proliferation, but this effect is indirect (Barrett, 2008). Exit from quiescence is physiologically coupled to larval growth and development via a nutritional stimulus (Britton, 1998). The Drosophila fat body performs many of the storage and endocrine functions of the vertebrate liver and acts as a sensor, coupling nutritional state to organismal growth. In response to dietary amino acids, the fat body secretes a mitogen that acts on the CNS to bring about neuroblast proliferation (Britton, 1998). This fat body-derived mitogen (FBDM) initiates cell growth in quiescent neuroblasts and promotes (or at least permits) cell-cycle re-entry (Britton, 1998). Yet the identity of the FBDM, the cell type upon which it acts, and the downstream pathway activated in neuroblasts have remained unknown (Chell, 2010).
Neuroblast entry into quiescence is governed intrinsically by the same transcription factor cascade that controls neuroblast temporal identity. This study has identified a population of surface glial cells that respond to the nutrition-dependent stimulus by expressing dILPs, and showns that the dInR/PI3K pathway is required by neuroblasts to exit quiescence in response to nutrition. Forced expression of dILPs in glia or activation of PI3K/Akt signaling in neuroblasts can drive neuroblast growth and proliferation in the absence of dietary protein and thus uncouple neuroblast reactivation from systemic nutritional control (Chell, 2010).
Cell growth and division are not strictly coupled in neuroblasts. In Drosophila Perlecan (dPerlecan) loss-of-function mutants, the majority of neuroblasts appear to increase in size but then remain G1 arrested. This suggested that a dedicated mitogen might exist to promote cell-cycle progression. Drosophila Activin-like peptides (ALPs; Zhu, 2008) are required for normal levels of neuroblast division in the larval brain and appear to be one such dedicated mitogen (Chell, 2010).
Perlecan is expressed by glia and forms part of the basement membrane that enwraps the CNS. Perlecan was proposed to modulate Drosophila FGF [Branchless (Bnl)], allowing it to act as a mitogen for neuroblasts. However, it now appears that the action of Bnl is indirect via a still to be identified cell type (Barrett, 2008). One possibility is that Bnl acts on glia to modulate the expression of other proteins, such as dILPs or ALPs, which then in turn act on neuroblasts directly. This study shows that expression of dILPs by glia leads to neuroblast reactivation in the absence of dietary protein; however, the number of mitoses falls short of that seen under normal dietary conditions. This could be explained by the absence of another nutritionally dependent mitogen. It will be of interest to see whether the glial expression of ALPs, like that of dILPs, relies on dietary protein (Chell, 2010).
In the larval CNS, neuroblasts and their progeny are completely surrounded by glial cell processes. If the interaction between neuroblasts and surrounding glia is disrupted by expression of a dominant-negative form of DE-cadherin, the mitotic activity of neuroblasts is severely reduced (Dumstrei, 2003). In the mammalian brain, glial cells are involved in a wide variety of processes, including axon guidance, synapse formation, and neuronal specification. Glial cells, with the extracellular matrix and vasculature, also make up the adult neural stem cell niche. Astrocytes have been shown to promote neural stem cell proliferation in culture and can express proproliferative factors such as FGF-2 and IGF-I. Thus, astrocytes are thought to be a key component of the niches that dynamically regulate neural stem cell proliferation in the adult brain (Chell, 2010).
This study has shown that Drosophila surface glia can transduce systemic signals and, by expressing dILP2 and dILP6, control neuroblast exit from quiescence. Glial cells also express dPerlecan and ana and are the source of the Activin-like peptides that have a direct mitogenic effect on neuroblasts. Thus, much like mammalian glial cells, Drosophila glial cells perform a number of the functions that define a niche and control the proliferation of neural stem cells (Chell, 2010).
Recent results suggest a role for IGF-1 in the control of neural stem cell division (Mairet-Coello et al., 2009). IGF-1 injection into rat embryonic brain results in a 28% increase in DNA content postnatally as a consequence of increased DNA synthesis and entry into S phase. Conversely, DNA synthesis and entry into S phase are decreased when the PI3K/Akt pathway is blocked. Furthermore, the loss of PTEN, the tumor suppressor and PI3K antagonist, enhances the exit from G0 of neural stem cells cultured from mouse embryonic cortex. It was suggested that a concomitant increase in cell size may push the cells to enter G1 (Chell, 2010).
This study shows, in vivo, that glial expression of insulin-like peptides activates the dInR/PI3K/Akt pathway in Drosophila neural stem cells and is responsible for their exit from quiescence. This pathway promotes cell growth and the transition from G0 to G1 and is also sufficient to promote G1-S and mitosis. Given that IGF-1 and the PI3K/Akt pathway can promote cell-cycle progression in vertebrate neural stem cells, this same pathway may regulate vertebrate neural stem cell reactivation in the same way as has been shown in this study for Drosophila (Chell, 2010).
The identity of the proposed FBDM, secreted by the fat body in response to dietary protein, remains unknown. However, explant CNS culture experiments demonstrated that the FBDM can act directly on the CNS to bring about neuroblast reactivation (Britton, 1998). This study has identified the surface glia as a key relay in the nutritional control of neuroblast proliferation. If the receptor protein(s) that controls glial dILP expression/secretion can be identified, then, by extension, it might be possible to identify the FBDM and approach a comprehensive understanding of how neural stem cell proliferation is coupled to nutrition and organism-wide growth (Chell, 2010).
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).
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).
Multicellular animals match costly activities, such as growth and reproduction, to the environment through nutrient-sensing pathways. The insulin/IGF signaling (IIS) pathway plays key roles in growth, metabolism, stress resistance, reproduction, and longevity in diverse organisms including mammals. Invertebrate genomes often contain multiple genes encoding insulin-like ligands, including seven Drosophila insulin-like peptides (DILPs). This study investigated the evolution, diversification, redundancy, and functions of the DILPs, combining evolutionary analysis, based on the completed genome sequences of 12 Drosophila species, and functional analysis, based on newly-generated knock-out mutations for all 7 dilp genes in D. melanogaster. Diversification of the 7 DILPs preceded diversification of Drosophila species, with stable gene diversification and family membership, suggesting stabilising selection for gene function. Gene knock-outs demonstrated both synergy and compensation of expression between different DILPs, notably with DILP3 required for normal expression of DILPs 2 and 5 in brain neurosecretory cells and expression of DILP6 in the fat body compensating for loss of brain DILPs. Loss of DILP2 increased lifespan and loss of DILP6 reduced growth, while loss of DILP7 did not affect fertility, contrary to its proposed role as a Drosophila relaxin. Importantly, loss of DILPs produced in the brain greatly extended lifespan but only in the presence of the endosymbiontic bacterium Wolbachia, demonstrating a specific interaction between IIS and Wolbachia in lifespan regulation. Furthermore, loss of brain DILPs blocked the responses of lifespan and fecundity to dietary restriction (DR) and the DR response of these mutants suggests that IIS extends lifespan through mechanisms that both overlap with those of DR and through additional mechanisms that are independent of those at work in DR. Evolutionary conservation has thus been accompanied by synergy, redundancy, and functional differentiation between DILPs, and these features may themselves be of evolutionary advantage (Grönke, 2010. Full text of article)
Metabolic adaptation to changing dietary conditions is critical to maintain homeostasis of the internal milieu. In metazoans, this adaptation is achieved by a combination of tissue-autonomous metabolic adjustments and endocrine signals that coordinate the mobilization, turnover, and storage of nutrients across tissues. To understand metabolic adaptation comprehensively, detailed insight into these tissue interactions is necessary. This study characterize the tissue-specific response to fasting in adult flies and identified an endocrine interaction between the fat body and liver-like oenocytes that regulates the mobilization of lipid stores. Using tissue-specific expression profiling, it was confirmed that oenocytes in adult flies play a central role in the metabolic adaptation to fasting. Furthermore, it was found that fat body-derived Drosophila insulin-like peptide 6 (dILP6) induces lipid uptake in oenocytes, promoting lipid turnover during fasting and increasing starvation tolerance of the animal. Selective activation of insulin/IGF signaling in oenocytes by a fat body-derived peptide represents a previously unidentified regulatory principle in the control of metabolic adaptation and starvation tolerance (Chatterjee, 2014).
Metabolic organs such as the liver and adipose tissue produce several peptide hormones that influence metabolic homeostasis. Fat bodies, the Drosophila counterpart of liver and adipose tissues, have been thought to analogously secrete several hormones that affect organismal physiology, but their identity and regulation remain poorly understood. Previous studies have indicated that microRNA miR-8, functions in the fat body to non-autonomously regulate organismal growth, suggesting that fat body-derived humoral factors are regulated by imiR-8. This study found that several putative peptide hormones known to have mitogenic effects are regulated by imiR-8 in the fat body. Most members of the imaginal disc growth factors and two members of the adenosine deaminase-related growth factors are up-regulated in the absence of imiR-8. Drosophila insulin-like peptide 6 (Dilp6) and Imaginal morphogenesis protein-late 2 (Imp-L2), a binding partner of Dilp, are also up-regulated in the fat body of miR-8 null mutant larvae. The fat body-specific reintroduction of miR-8 into the miR-8 null mutants revealed six peptides that showed fat-body organ-autonomous regulation by miR-8. Amongst them, only Imp-L2 was found to be regulated by U-shaped, the miR-8 target for body growth. However, a rescue experiment by knockdown of Imp-L2 indicated that Imp-L2 alone does not account for miR-8's control over the insect's growth. These findings suggest that multiple peptide hormones regulated by miR-8 in the fat body may collectively contribute to Drosophila growth (Lee, 2014).
Search PubMed for articles about Drosophila Ilp6
Aguila, J. R., Suszko, J., Gibbs, A. G., and Hoshizaki, D. K. (2007). The role of larval fat cells in adult Drosophila melanogaster. J. Exp. Biol. 210: 956-963. PubMed ID: 17337708
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 ID: 22745312
Barrett, A.L., Krueger, S., and Datta, S. (2008). Branchless and Hedgehog operate in a positive feedback loop to regulate the initiation of neuroblast division in the Drosophila larval brain. Dev. Biol. 317: 234-245. PubMed ID: 18353301
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date revised: 2 December 2018
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