InteractiveFly: GeneBrief

Insulin-like peptide 6: Biological Overview | References

BIOLOGICAL OVERVIEW

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).

A fat body-derived IGF-like peptide regulates postfeeding growth in Drosophila

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).

Deletion of Drosophila insulin-like peptides causes growth defects and metabolic abnormalities

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).

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

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

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

Drosophila 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).

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

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

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

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

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

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

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

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

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

Molecular evolution and functional characterization of Drosophila insulin-like peptides

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)

Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes

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).

MicroRNA miR-8 regulates multiple growth factor hormones produced from Drosophila fat cells

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).

Regulatory roles of Drosophila Insulin-Like Peptide 1 (DILP1) in metabolism differ in pupal and adult stages

The insulin/IGF-signaling pathway is central in control of nutrient-dependent growth during development, and in adult physiology and longevity. Eight insulin-like peptides (DILP1-8) have been identified in Drosophila, and several of these are known to regulate growth, metabolism, reproduction, stress responses, and lifespan. However, the functional role of DILP1 is far from understood. Previous work has shown that dilp1/DILP1 is transiently expressed mainly during the pupal stage and the first days of adult life. The role of dilp1 in the pupa, as well as in the first week of adult life, was studied, and some comparisons were made to dilp6 that displays a similar pupal expression profile, but is expressed in fat body rather than brain neurosecretory cells. Mutation of dilp1 diminishes organismal weight during pupal development, whereas overexpression increases it, similar to dilp6 manipulations. No growth effects of dilp1 or dilp6 manipulations were detected during larval development. It was next show that dilp1 and dilp6 increase metabolic rate in the late pupa and promote lipids as the primary source of catabolic energy. Effects of dilp1 manipulations can also be seen in the adult fly. In newly eclosed female flies, survival during starvation is strongly diminished in dilp1 mutants, but not in dilp2 and dilp1/dilp2 mutants, whereas in older flies, only the double mutants display reduced starvation resistance. Starvation resistance is not affected in male dilp1 mutant flies, suggesting a sex dimorphism in dilp1 function. Overexpression of dilp1 also decreases survival during starvation in female flies and increases egg laying and decreases egg to pupal viability. In conclusion, dilp1 and dilp6 overexpression promotes metabolism and growth of adult tissues during the pupal stage, likely by utilization of stored lipids. Some of the effects of the dilp1 manipulations may carry over from the pupa to affect physiology in young adults, but the data also suggest that dilp1 signaling is important in metabolism and stress resistance in the adult stage (Liao, 2020).

The insulin/IGF signaling (IIS) pathway plays a central role in nutrient-dependent growth control during development, as well as in adult physiology and aging. More specifically, in mammals, insulin, IGFs, and relaxins act on different types of receptors to regulate metabolism, growth, and reproduction. This class of peptide hormones has been well conserved over evolution and therefore the genetically tractable fly Drosophila is an attractive model system for investigating IIS mechanisms. Eight insulin-like peptides (DILP1-8), each encoded on a separate gene, have been identified in Drosophila. The genes encoding these DILPs display differential temporal and tissue-specific expression profiles, suggesting that they have different functions. Specifically, DILP1, 2, 3, and 5 are mainly expressed in median neurosecretory cells located in the dorsal midline of the brain, designated insulin-producing cells (IPCs). The IPC-derived DILPs can be released into the open circulation from axon terminations in the corpora cardiaca, the anterior aorta, the foregut, and the crop. Genetic ablation of the IPCs reduces growth and alters metabolism, and results in increased resistance to several forms of stress and prolongs lifespan (Liao, 2020).

The functions of the individual DILPs produced by the IPCs may vary depending on the stage of the Drosophila life cycle. Already, the temporal expression patterns hint that DILP1-3 and 5 play different roles during development. Thus, whereas DILP2 and 5 are relatively highly expressed during larval and adult stages, DILP1 and 6 are almost exclusively expressed during pupal stages under normal conditions (Liao, 2020).

DILP1 is unique among the IPC-produced peptides since it can be detected primarily during the pupal stage (a non-feeding stage) and the first few days of adult life when residual larval/pupal fat body is present. Furthermore, in female flies kept in adult reproductive diapause, where feeding is strongly reduced, dilp1 is different from the other insulin-like peptides tested (Liao, 2020). DILP1 is unique among the IPC-produced peptides since it can be detected primarily during the pupal stage (a non-feeding stage) and the first few days of adult life when residual larval/pupal fat body is present. Furthermore, in female flies kept in adult reproductive diapause, where feeding is strongly reduced, dilp1/DILP1 expression is also high (Liu, 2016). The temporal expression profile of dilp1/DILP1 resembles that of dilp6/DILP6 although the latter peptide is primarily produced by the fat body, not IPCs. Since DILP6 was shown to regulate growth of adult tissues during pupal development, it was asked whether also DILP1 plays a role in growth control. It is known that overexpression of several of the DILPs is sufficient to increase body growth through an increase in cell size and cell number, and especially DILP2 produces a substantial increase in body weight. In contrast, not all single dilp mutants display a decreased body mass. The dilp1, dilp2, and dilp6 single mutants display slightly decreased body weight, whereas the dilp3, dilp4, dilp5, and dilp7 single mutants display normal body weight. However, a triple mutation of dilp2, 3, and 5 causes a drastically reduced body weight, and a dilp1-4,5 mutation results in a further reduction. Note that several of the above studies do not show bona fide effects on cell or organismal growth (e.g., volume or cell numbers/sizes); they only provide body mass data (Liao, 2020).

There is a distinction between how DILPs act in growth regulation. DILPs other than DILP1 and DILP6 promote growth primarily during the larval stages (both feeding and wandering stages) when their expression is high. This nutrient-dependent growth is relatively well-understood and is critical for production of the steroid hormone ecdysone and thereby developmental timing and induction of developmental transitions such as larval molts and pupariation. The growth in the pupal stage, which primarily affects imaginal discs and therefore adult tissues, is far less studied. This study investigated the role of dilp1/DILP1 in growth regulation in Drosophila in comparison to dilp6/DILP6. For this, both bona fide size of body and/or wings were determined and wet weights were provided, and thus it was possible to distinguish between growth and increase of body mass. Mutation of dilp1 diminishes body weight (but not body size), whereas ectopic dilp1 expression promotes organismal growth by increasing both weight and size during the pupal stage, similar to dilp6. Thus, we cannot unequivocally show a role of dilp1 in organismal growth, but it does regulate body mass, suggesting that dilp1 affects metabolism and energy stores. Determination of metabolic rate (MR) and respiratory quotient (RQ) as well as triacylglyceride (TAG) levels during late pupal development provides evidence that dilp1 and dilp6 increase the MR and that the associated increased metabolic cost is fueled by increased lipid catabolism (Liao, 2020).

Since dilp1/DILP1 levels are high the first week of adult life, the role of dilp1 mutation and overexpression on early adult physiology was determined, including metabolism stress resistance and fecundity. Interestingly, the newly eclosed dilp1 mutant flies are less resistant to starvation than controls and dilp2 mutants. Thus, dilp1 acts differently from other dilps for which it has been shown that reduced signaling increases survival during starvation. Also, early egg laying and female fecundity are affected by dilp1 overexpression, and in general, dilp1 manipulations produce more prominent effects in female flies (Liao, 2020).

Taken together, these data suggest that ectopic expression of dilp1/DILP1 promotes growth of adult tissues during the pupal stage, and that this process mainly utilizes stored lipids to fuel the increased MR. The DILP1 signaling also affects the metabolism in the young adult fly, and sex dimorphic effects of altered signaling on stress responses and fecundity were seen (Liao, 2020).

This study shows that dilp1 gain of function stimulates adult tissue growth and increases metabolic rate (MR) during the pupal stage, and also affects adult physiology, especially during the first days of adult life. These stages correspond to the time when dilp1 is normally expressed. The gain of function experiments in this study suggest that the developmental role of ectopic dilp1 could be similar to that of dilp6, namely, to stimulate growth of adult tissues during pupal development. It was furthermore shown that in the adult fly, dilp1 is upregulated during starvation and genetic gain and loss of function of dilp1 signaling diminishes the flies' survival under starvation conditions in a sex-specific manner. These novel findings, combined with previous data that demonstrated high levels of dilp1 during adult reproductive diapause and the role of dilp1 as a pro-longevity factor during aging, suggest a wide-ranging importance of this signaling system. Not only does dilp1 expression correlate with stages of non-feeding (or reduced feeding), these stages are also associated with lack of reproductive activity and encompass the pupa, newly eclosed flies, and diapausing flies. Under normal conditions, the transient expression of dilp1/DILP1 during the first few days of adult life may be associated with a metabolic transition (remodeling from larval to adult fat body) and the process of sexual maturation (gonad growth and differentiation). The data also suggest that dilp1 affects physiology more prominently in young female flies than in males, which might be associated with ovary maturation (Liao, 2020).

It is also interesting to note that the diminished starvation resistance in dilp1 and dilp1/dilp2 mutants is opposite to the phenotype seen after IPC ablation, mutation of dilp1-4, or diminishing IIS by other genetic interventions. Thus, in recently eclosed flies, dilp1 appears to promote starvation resistance rather than diminishing it. Furthermore, the decreased survival during starvation in female dilp1 mutants is the opposite of that shown in dilp6 mutants, indicating that dilp1 action is different from the other insulin-like peptides tested (Liao, 2020).

In Drosophila, the final body size is determined mainly by nutrient-dependent hormonal action during the larval feeding stage. However, some regulation of adult body size can also occur after the cessation of the larval feeding stage, and this process is mediated by dilp6 acting on adult tissue growth in the pupa in an ecdysone-dependent manner. This is likely a mechanism to ensure growth of adult tissues if the larva is exposed to shortage of nutrition during its feeding stage. The current findings suggest that dilp1 can function as another regulator of growth during the pupal stage. Overexpression of dilp1 promotes organismal growth in the pupa, probably at the cost of stored nutrients derived from the larval feeding stage. This is supported by respiratory quotient (RQ) data that clearly show a shift from mixed-energy substrate metabolism in control flies toward almost pure lipid catabolism at the end of pupal development in the dilp1 overexpression flies (also seen for dilp6 gain of function in these experiments). Furthermore, triacylglycerol (TAG) (but not carbohydrate) levels in dilp1 overexpression pupae were clearly decreased, which likely reflects the shift in catabolic energy substrate also seen in the RQ using respirometry. It should be noted that insects predominantly use lipids as fuel during metamorphosis and dilp1 overexpression increases lipid catabolism. This study hence suggests that dilp1 can parallel dilp6 in balancing adult tissue growth and storage of nutrient resources during pupal development. This is interesting since dilp6 is an IGF-like peptide that is produced in the nutrient sensing fat body, whereas the source of the insulin-like dilp1 is the brain IPCs (Liao, 2020).

In contrast to the dilp1 gain of function, the experiments with dilp1 mutant flies did not show a clear effect on adult body growth, only a decrease in weight. Is this a result of compensation by other DILPs? Previous work showed that young adult dilp1 mutant flies display increased dilp6 and vice versa, suggesting feedback between these two peptide hormones in adults. During the pupal stage, this feedback appears less prominent in dilp1 mutants and no effects were detected on (dilp2, dilp3), or dilp6 levels. Furthermore, overexpression of dilp1 in fat body or IPCs has no effect on pupal levels of dilp2 and dilp6. Thus, at present, there is no evidence for compensatory changes in other dilps/DILPs in pupae with dilp1 manipulations. However, under normal conditions (in wild-type pupae), dilp6 levels are far higher than those of dilp1, which could buffer the effects of changes in dilp1 signaling (Liao, 2020).

DILPs and IIS are involved in modulating responses to starvation, desiccation, and oxidative stress in Drosophila. Flies with ablated IPCs or genetically diminished IIS display increased resistance to several forms of stress, including starvation . Conversely, overexpression of dilp2 increases mortality in Drosophila. This study found that young dilp1 mutant flies displayed diminished starvation resistance. In both recently eclosed and 3-day-old flies, mutation of dilp1 decreased survival during starvation (but not in 6- to 7-day-old flies) (Liao, 2020).

Action of dilp1 in the adult fly is also linked to reproductive diapause in females, where feeding is strongly reduced, and both peptide and transcript are upregulated. Related to this, dilp1 mRNA was found to upregulated during starvation in 12-day-old flies. Furthermore, it was shown that expression of dilp1 (dilp1 rescue) increases lifespan in dilp1/dilp2 double mutants, suggesting that loss of dilp2 induces dilp1 as a factor that promotes longevity. Thus, dilp1 activity is beneficial also during adult life, even though its expression under normal conditions is very low. This pro-longevity effect of dilp1 is in contrast to dilp2, 3, and 5 and the mechanisms behind this effect are of great interest to unveil (Liao, 2020).

A previous study showed that in wild-type (Canton S) Drosophila, DILP1 expression in young adults is sex-dimorphic with higher levels in females. In line with this, starvation resistance in young flies is diminished only in female dilp1 mutant and dilp1 overexpression flies. Thus, taken together, previous work showed that dilp1 displays a sex-specific expression and this study shows female-specific function in young adult Drosophila. It is tempting to speculate that the more prominent role of dilp1 in female flies is linked to metabolism associated with reproductive physiology and early ovary maturation, which is also reflected in the dilp1 upregulation during reproductive diapause. In fact, this study shows that egg-laying increased after dilp1 overexpression, and an earlier study demonstrated a decreased egg laying in dilp1 mutant flies. Part of the sex dimorphic effects on body weight of young adults after dilp1 manipulations might be a result of a differential role of dilp1 in water homeostasis (Liao, 2020).

This study shows that IPC-derived dilp1 displays several similarities to the fat body-produced dilp6, including temporal expression pattern, growth promotion, effects on adult stress resistance and lifespan. Additionally, dilp1 may play a role in regulation of nutrient utilization and metabolism during the first few days of adult life, especially in females. At this time, larval fat body is still present and utilized as energy fuel/nutrient store and this source also contributes to egg development. Curiously, there is a change in the action of DILP1 between the pupal and adult stages from being able to stimulate growth (agonist of dInR, like DILP6) in pupae, to acting in a manner opposite to DILP2, DILP6, and other DILPs in adults in regulation of lifespan and stress responses. Only one dInR is known so far (excluding the G protein-coupled receptors for the relaxin-like DILP7 and DILP8). Thus, the mechanisms behind this apparent switch in function of DILP1 signaling remain an open question. One possibility is that DILP1 acts via different signaling pathways downstream the dInR in pupae and adults. An obvious difference between these two stages is the presence of larval-derived fat body in the pupa and during the first few days of adults and its replacement by functional adult fat body in later stages. Perhaps dInR-mediated action differs in these types of fat body when activated by DILP1. Another possibility is stage-specific expression of insulin/IGF-binding proteins such as SDR, ALS, and Imp-L2 that could affect the activity of DILP1 in particular (Liao, 2020).

In the future, it would be interesting to investigate whether DILP1 acts differently on larval/pupal and adult fat body, or act on different downstream signaling in the two stages of the life cycle. Also, the possibility that dilp1 and dilp6 interact to regulate growth and metabolism in Drosophila is worth pursuing (Liao, 2020).

Regenerative neurogenic response from glia requires insulin-driven neuron-glia communication

Understanding how injury to the central nervous system induces de novo neurogenesis in animals would help promote regeneration in humans. Regenerative neurogenesis could originate from glia and glial neuron-glia antigen-2 (NG2) may sense injury-induced neuronal signals, but these are unknown. This study used Drosophila to search for genes functionally related to the NG2 homologue kon-tiki (kon), and identified Islet Antigen-2 (Ia-2), required in neurons for insulin secretion. Both loss and over-expression of ia-2 induced neural stem cell gene expression, injury increased ia-2 expression and induced ectopic neural stem cells. Using genetic analysis and lineage tracing, this study demonstrated that Ia-2 and Kon regulate Drosophila insulin-like peptide 6 (Dilp-6) to induce glial proliferation and neural stem cells from glia. Ectopic neural stem cells can divide, and limited de novo neurogenesis could be traced back to glial cells. Altogether, Ia-2 and Dilp-6 drive a neuron-glia relay that restores glia and reprogrammes glia into neural stem cells for regeneration (Harrison, 2021).

The central nervous system (CNS) can regenerate after injury in some animals, and this involves de novo neurogenesis. Newly formed neurons integrate into functional neural circuits, enabling the recovery of function and behaviour, which is how CNS regeneration is measured. The human CNS does not regenerate after injury. However, in principle it could, as we continue to produce new neurons throughout life that integrate into functional circuits. Through understanding the molecular mechanisms underlying natural regenerative neurogenesis in animals, it might be possible to provoke de novo neurogenesis in the human CNS to promote regeneration after damage or neurodegenerative diseases. Regenerative neurogenesis across animals may reflect an ancestral, evolutionarily conserved genetic mechanism, which manifests itself to various degrees in regenerating and non-regenerating animals. Accordingly, it may be possible to discover molecular mechanisms of injury-induced neurogenesis in the fruit-fly Drosophila, which is a powerful genetic model organism (Harrison, 2021).

Regenerative neurogenesis could occur through activation of quiescent neural stem cells, de-differentiation of neurons or glia, or direct conversion of glia to neurons. Across many regenerating animals, new neurons originate mostly from glial cells. In the mammalian CNS, radial glial cells behave like neural stem cells to produce neurons during development. Remarkably, whereas NG2-glia (also known as oligodendrocyte progenitor cells, OPCs) produce only glia (oligodendrocytes and astrocytes) in development, they can also produce neurons in the adult and upon injury, although this remains controversial. Discovering the molecular mechanisms of a neurogenic response of glia is of paramount urgency (Harrison, 2021).

NG2-glia are progenitor cells in the adult human brain, constituting 5-10% of total CNS cells, and remain proliferative throughout life. In development, NG2-glia are progenitors of astrocytes, OPCs, and oligodendrocytes, but postnatally and upon injury they can also produce neurons. They can also be directly reprogrammed into neurons that integrate into functional circuits. The diversity and functions of NG2-glia are not yet fully understood, but they are particularly close to neurons. They receive and respond to action potentials generating calcium signals, they monitor and modulate the state of neural circuits by regulating channels and secreting chondroitin sulphate proteoglycan perineural nets, and they also induce their own proliferation to generate more NG2-glia, astrocytes that sustain neuronal physiology, and oligodendrocytes that enwrap axons. NG2-glia have key roles in brain plasticity, homeostasis, and repair in close interaction with neurons, but to what extent this depends on the NG2 gene and protein, is not known (Harrison, 2021).

NG2 (also known as chondroitin sulphate proteoglycan 4, CSPG4) is expressed by NG2-glia and pericytes, but not by oligodendrocytes, neurons, or astrocytes. NG2 is a transmembrane protein that can be cleaved upon neuronal stimulation to release a large secreted extracellular domain and an intracellular domain. The intracellular domain (ICD, NG2ICD) is mostly cytoplasmic, and it induces protein translation and cell cycle progression (Nayak, 2018). NG2ICD lacks a DNA binding domain and therefore does not function as a transcription factor, but it has a nuclear WW4 domain and nuclear localisation signals and can regulate gene expression. It is thought that NG2 functions as a receptor, triggering nuclear signalling in response to ligands or partners (Sakry, 2014; Sakry and Trotter, 2016). NG2 protein is abundant in proliferating NG2-glia and glioma. It is also required for OPC proliferation and migration in development and in response to injury. Given the close relationship of NG2-glia with neurons, it is anticipated that key partners of NG2 are produced from neurons, but these remain largely unknown (Harrison, 2021).

The fruit-fly Drosophila is particularly powerful for discovering novel molecular mechanisms. The Drosophila NG2 homologue is called kon-tiki (kon) or perdido. Kon functions in glia, promotes glial proliferation and glial cell fate determination in development and upon injury, and promotes glial regeneration and CNS injury repair. Kon works in concert with the receptor Notch and the transcription factor Prospero (Pros) to drive the glial regenerative response to CNS injury. It is normally found in low levels in the larval CNS, but injury induces a Notch-dependent increase in kon expression in glia. Together, Notch signalling and Kon induce glial proliferation. Kon also initiates neuropile glial differentiation and pros expression, and Pros maintains glial cell differentiation. This glial regenerative response to injury is homeostatic and time-limited, as two negative feedback loops halt it: Kon represses Notch, and Pros represses kon expression, preventing further cell division. The relationship between these genes is also conserved in the mouse, where the homologue of pros, Prox1, is expressed together with Notch1 in NG2-glia. Following cell division, Prox1 represses NG2-glia proliferation and promotes oligodendrocyte differentiation. Together, Notch, Kon, and Pros form a homeostatic gene network that sustains neuropile glial integrity throughout life and drives glial regeneration upon injury. As Kon is upregulated upon injury and provokes glial proliferation and differentiation, it is the key driver of the glial regenerative response to CNS injury (Harrison, 2021).

A critical missing link to understand CNS regeneration was the identification of neuronal partners of glial NG2/Kon that could induce regenerative neurogenesis. Injury to the Drosophila larval CNS also resulted in spontaneous, yet incomplete, repair of the axonal neuropile. This strongly suggested that injury might also induce neuronal events, such as axonal regrowth or generation of new neurons. Thus, this study asked whether Kon may interact with neuronal factors that could contribute to regenerative neurogenesis after injury. Relay of insulin signalling involving neuronal Ia-2 and glial Kon drives in vivo reprogramming of neuropile glia into neural stem cells (Harrison, 2021).

NG2-glia are abundant progenitor cells present throughout life in the adult human brain and can respond to injury. Thus, they are the ideal cell type to manipulate to promote regeneration. However, whether NG2-glia can give rise to neurons is highly debated, and potential mechanisms remained unknown. Using Drosophila in vivo functional genetic analysis this study has identified neuronal Ia-2 as a genetic interactor of the NG2 homologue Kon and shows that it can induce a neurogenic response from glial cells via insulin signalling (Harrison, 2021).

Evidence is provided that Ia-2, Kon, and Dilp-6 induce a regenerative neurogenic response from glia (Ia-2 and Dilp-6 drive a regenerative neurogenic response to central nervous system (CNS) injury). In the un-injured CNS, Kon and Ia-2 are restricted to glia and neurons, respectively (Ia-2 and Dilp-6 drive a regenerative neurogenic response to central nervous system (CNS) injury). Ia-2 is required for neuronal Dilp-6 secretion, Dilp-6 is produced by some neurons and mostly glia, and its production depends mostly on Kon regulated glia. Alterations in Ia-2 levels, increased Dilp-6, and concerted activation of Ras or PI3Kinase downstream of insulin signalling induced ectopic neural stem cells from glia. Both loss and gain of ia-2 function induced ectopic Dpn cells. Ia-2 depends on Pros and in turn negatively regulates Pros. Pros controls the switch from neural stem cell to progenitor state. In this way, cell-cell interactions involving Ia-2 can influence neural progenitor cell fate. ia-2 loss of function would also cause a decrease in Dilp-6 secretion from neurons, but not from glia, as kon mRNA levels were unaffected, and dilp-6 expression depends mostly on glial kon. As neuronal Ia-2 and glial Kon mutually exclude each other, perhaps loss of ia-2 function might increase kon-dependent Dilp-6 production. As Ia-2 is required for Dilp-6 secretion, ia-2 GOF would increase Dilp-6 release triggering the Dilp-6 amplification loop. Conceivably, either way Dilp-6 increased and this induced Dpn. Upon injury, levels of kon and ia-2 expression increased. Ia-2 drives secretion of Dilp-6 from neurons, Dilp-6 is received by glia, and a positive feedback amplification loop drives the further Kon and InR dependent production of Dilp-6 from cortex glia. Dilp-6 can then both promote glial proliferation to generate more glia and induce the neural stem cell marker Dpn in neuropile glia -- the subset known as 'Drosophila astrocytes' and midline glia. Ectopic Dpn+ cells were induced from glia both upon injury and genetic manipulation of Ia-2, Dilp-6, Ras, and PI3Kinase. Importantly, these glial-derived neural stem cells could divide, as revealed by the S-phase marker PCNA-GFP and the mitotic marker pH3, and could generate neurons, albeit to a rather limited extent. Altogether, Dilp-6 is relayed from neurons to cortex and then to neuropile glia. This neuron-glia communication relay could enable concerted glio- and neuro-genesis, matching interacting cell populations for regeneration. Interestingly, Dilp-6 is also involved in non-autonomous relays between distinct CNS cell populations to activate neural stem cells and induce neuronal differentiation in development (Harrison, 2021).

This study has demonstrated that ectopic neural stem cells originate from glia. Regenerative neurogenesis could occur via direct conversion of glia into neurons, glial de-differentiation, or neuronal de-differentiation. Neuronal de-differentiation occurs both in mammals and in Drosophila. However, in most animals, neural stem cells in the adult CNS and upon injury are generally distinct from developmental ones, and can originate from hemocytes, but most often, glial cells. In the mammalian brain, radial glia in the hippocampus respond to environmental challenge by dividing asymmetrically to produce neural progenitors that produce neurons; and astrocytes and NG2-glia can generate neurons, particularly in response to stroke, excitoxic injury, and genetic manipulations. Furthermore, genetic manipulation can lead to the direct conversion of NG2-glia into neurons. The findings that Dilp-6 and InR signalling can induce dpn expression are reminiscent of their functions in the induction of neural stem cells from quiescent progenitors in development. However, the Dpn+ cells induced upon injury and after development are distinct from the developmental neural stem cells normally induced by Dilp-6 in multiple ways. Firstly, in injuries carried out in third instar larvae, the induced neural stem cells were more numerous than normal neural stem cells. Secondly, in injuries carried out late in wandering larvae, Dpn+ cells were found after normal developmental neural stem cells have been eliminated through apoptosis. Thirdly, Dpn+ cells were found in dorsal ectopic locations not normally occupied by developmental neural stem cells. In all injury and genetic manipulation experiments involving overexpression of either ia-2, dilp-6, or PI3K, ectopic Dpn+ cells were located along the midline and surrounding the neuropile, in positions normally occupied by glia. Remarkably, concerted overexpression of ras and dilp-6 induced Dpn in potentially all glial cells and more, consistently with further Dpn+ cell proliferation. Consistent with the current findings, ectopic neuroblasts were also observed upon co-expression of activated rasV12 and knock-down of PTEN in glia, within glioma models in Drosophila. This study has demonstrated that ectopic Dpn+ originated from glia, most particularly neuropile glia (midline glia and 'Drosophila astrocytes'). Firstly, ectopic Dpn+ cells did not have Ia-2YFP, which is expressed in all neurons. Secondly, overexpression of ia-2 or dilp-6, alone or in combination with ras and PI3K, in glia dramatically increased Dpn levels, meaning that insulin signalling induces dpn expression in glia. Thirdly, ectopic Dpn+ cells surrounding the neuropile occupied positions of astrocytes and had the pan-glial marker Repo, and Repo- Dpn+ along the midline had the midline glia marker Wrp. Fourthly, the glial origin of the ectopic Dpn+ cells was demonstrated using two cell-lineage tracing methods (G-TRACE and glial activation of the actin promoter) whereby the expression initiated from the glia repo promoter was turned permanent despite cell state transitions. Consistently with these findings, TRAP-RNA analysis of the normal third instar larva revealed expression of dpn and multiple genes involved in neuroblast polarity, asymmetric cell division, neuroblast proliferation, and neurogenesis in glia. And single cell RNAseq analysis of the larval CNS revealed that in normal larvae some Repo+ glial cells can express dpn, or other neuroblast markers like wor and ase. The current findings show that basal or potential expression of neuroblast genes in glia is switched on and amplified by insulin signalling. It is concluded that Ia-2 and Dilp-6 could reprogramme glial cells in vivo into neural stem cells (Harrison, 2021).

The data showed that the ectopic ia-2 and dilp-6 induced neural stem cells could divide and generate neurons. In fact, concomitant overexpression of dilp-6 and PI3K, and most prominently dilp-6 and ras, dramatically increased Dpn+ cell number. Dilp-6 induced glial-derived Dpn+ cells could express the S-phase marker PCNA-GFP, and Ia-2 induced Wrp+ Dpn+ cells that were pH3+ in mitosis. No mitotic cells surrounding the neuropile were detected, but mitosis is brief, and could have easily been missed. The Dilp-6 induced ectopic Dpn+ cells could generate neurons that could be traced with GFP expression from their glial origin. Thus, ectopic neural stem cells induced by Dilp-6 can divide and produce neuronal progeny cells. However, the clusters of GFP+ cells originating from the in vivo reprogrammed glial cells were rather small, indicating that although neurogenesis was possible in late larvae, it was extremely constrained. This could be due to the fact that in the third instar larva, time is rather limited by pupariation. Injury and genetic manipulation in late larvae may not allow sufficient time for cell lineages to progress, before pupariation starts. Pupariation and metamorphosis bring in a different cellular context, which could interfere with regenerative neuronal differentiation. Alternatively, Ia-2 and Dpn may not be sufficient to carry neurogenesis through either. For instance, gain of ia-2 function resulted only in Dpn+ but not Pros+ or Eve+ cells, suggesting that Ia-2 and Dpn are not sufficient for neuroblasts to progress to GMCs and neurons. In fact, ectopic Dpn+ cells still had Repo. Furthermore, other ectopic neuroblast markers, such as Wor or Ase were not detected in glia. Nevertheless, RNA seq data revealed expression of neuroblast markers, including dpn, wor, and ase in some glia in normal larval CNS, meaning they could potentially be further regulated. Still, to generate neurons, glia may not only require the expression of neural stem cell markers like dpn, but also perhaps receive other yet unknown signals. In mammals, injury creates a distinct cellular environment that prompts glial cells to generate different cell types than in the un-injured CNS. For instance, elevated Sox-2 is sufficient to directly reprogramme NG2-glia into neurons, but only upon injury. Whereas during normal development NG2-glial cells may only produce oligodendrocyte lineage cells, upon injury they can also produce astrocytes and neurons. This suggests that there are injury-induced cues for neuronal differentiation. In the future, it will be compelling to find out what signals could enhance neurogenesis from glial cells reprogrammed in vivo by insulin signalling (Harrison, 2021).

This work has revealed a novel molecular mechanism driving a regenerative neurogenic response from glia, involving Kon/NG2 and insulin signalling. Ia-2 induces an initial secretion of Dilp-6 from neurons, Dilp-6 is received by glia, and a positive feedback loop amplifies the Kon-dependent production of Dilp-6 by cortex glia, Dilp-6 is then relayed to neuropile glia, resulting in the in vivo reprogramming of glial cells into neural stem cells. This mechanism can induce both glial regeneration and neural stem cells from glia, potentially also neurons, matching interacting neuronal and glial cell populations. The incidence of neuropile glia conversion to Dpn+ cells was variable, meaning the process is stochastic. However, all glia converted when activated Ras or PI3K were combined with Dilp-6, meaning levels of insulin signalling matter. Such a mechanism may also operate in mammals. In fact, Ia-2 has universal functions in dense core vesicles to release insulin. Insulin-like growth factor 1 (IGF-1) induces the production of astrocytes, oligodendrocytes, and neurons from progenitor cells in the adult brain, in response to exercise. The transcription factor Sox-2 that can switch astrocytes to neural stem cells and produce neurons is a downstream effector of InR/AKT signalling). NG2 also interacts with downstream components of the InR signalling pathway (e.g., PI3K-Akt-mTOR) to promote cell cycle progression and regulate the expression of its downstream effectors in a positive feedback loop. Together, all of these findings indicate that Ia-2, NG2/Kon, and insulin signalling have a common function across animals in reprogramming glial cells into becoming neural stem cells (Harrison, 2021).

Intriguingly, dpn was mostly induced in neuropile associated glial cells and was only induced in other glial types with overexpression of active RasV12 together with Dilp-6. Thus, perhaps prominently neuropile glia have neurogenic potential. Of the neuropile glia, Drosophila 'astrocytes' and midline glia express Notch, pros, and kon, as well as InR. The cells frequently called 'astrocytes' share features with mammalian NG2-glia. In mammals, the combination of Notch1, Prox1, and NG2 is unique to NG2-glia and is absent from astrocytes. Perhaps Ia-2 and Dilp-6 can only induce neural stem cells from NG2-like glia bearing this combination of factors. Notch activates glial proliferation and kon expression in Drosophila, and in the mammalian CNS, Notch promotes NG2-glia proliferation and maintains the progenitor state. In Drosophila, Notch and Pros also regulate dpn expression: Notch activates dpn expression promoting stemness, and Pros inhibits it, promoting transition to GMC and neuron. Thus, only glial cells with Notch and Pros may be poised to modulate stemness and neuronal differentiation. This study showed that InR is expressed in neuropile glia, which was confirmed by publically available single cell RNAseq data. Insulin signalling represses FoxO, which represses dpn, and thus ultimately activates dpn expression. As Notch and insulin signalling positively regulate dpn expression, and injury induces a Notch-dependent upregulation of Kon, which enables dilp-6 expression, and of Ia-2, which secretes Dilp-6, the data indicate that Notch-Kon/NG2-insulin synergy triggers the activation of dpn expression. Importantly, no evidence was found that Kon functions in neural stem cells. Thus, perhaps induced neural stem cells can generate only glia from daughter cells that inherit Kon, on which Repo and glial cell fate depend, or generate neurons, from daughter cells that lack Kon, but have Pros, on which Ia-2 depends. Thus, upon injury, Notch, Pros, Kon/NG2, Ia-2, and insulin signalling function together to enable the regenerative production of both glial cells and neural stem cells from glia. Intriguingly, developmental neural stem cells are thought to be eliminated through upregulation of Pros, induction of cell cycle exit, and terminal differentiation into glia. The current findings imply that such termination may not be final (Harrison, 2021).

To conclude, a neuron-glia communication relay involving Ia-2, Dilp-6, Kon, and InR is responsible for the induction of neural stem cells from glia, their proliferation, and limited neurogenesis. Neuronal Ia-2 and Dilp-6 trigger two distinct responses in glia: (1) in cortex glial cells, insulin signalling boosts Kon-dependent amplification of Dilp-6, glial proliferation, and glial regeneration. (2) In neuropile-associated NG2-like glial cells, insulin signalling unlocks a neurogenic response, inducing neural stem cell fate. As a result, these genes can drive the production of both glial cells and neurons after injury, enabling the matching of interacting cell populations, which is essential for regeneration (Harrison, 2021).

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

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

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: 2149-2158. PubMed ID: 9570778

Brogiolo, W., Stocker, H., Ikeya, T., Rintelen, F., Fernandez, R., and Hafen, E. (2001). An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control. Curr. Biol. 11: 213-221. PubMed ID: 11250149

Chatterjee, D., Katewa, S. D., Qi, Y., Jackson, S. A., Kapahi, P. and Jasper, H. (2014). Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes. Proc Natl Acad Sci U S A 111(50):17959-64. PubMed ID: 25472843

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

Colombani, J., Raisin, S., Pantalacci, S., Radimerski, T., Montagne, J., and Léopold, P. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114: 739-749. PubMed ID: 14505573

Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, C., Carre, C., Noselli, S., and Léopold, P. (2005). Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science 310: 667-670. PubMed ID: 16179433

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

Géminard, C., Rulifson, E.J., and Léopold, P. (2009). Remote control of insulin secretion by fat cells in Drosophila. Cell Metab. 10: 199-207. PubMed ID: 19723496

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

Harrison, N. J., Connolly, E., Gascon Gubieda, A., Yang, Z., Altenhein, B., Losada Perez, M., Moreira, M., Sun, J. and Hidalgo, A. (2021). Regenerative neurogenic response from glia requires insulin-driven neuron-glia communication. Elife 10. PubMed ID: 33527895

Lee, G. J., Jun, J. W. and Hyun, S. (2014). MicroRNA miR-8 regulates multiple growth factor hormones produced from Drosophila fat cells. Insect Mol Biol [Epub ahead of print]. PubMed ID: 25492518

Liao, S., Post, S., Lehmann, P., Veenstra, J. A., Tatar, M. and Nassel, D. R. (2020). Regulatory roles of Drosophila Insulin-Like Peptide 1 (DILP1) in metabolism differ in pupal and adult stages. Front Endocrinol (Lausanne) 11: 180. PubMed ID: 32373064

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

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

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 ID: 18505882

Nakae, J., Kido, Y. and Accili, D. (2001). Distinct and overlapping functions of insulin and IGF-I receptors. Endocr. Rev. 22: 818-835. PubMed ID: 11739335

Okamoto, N., et al. (2009a). An ecdysteroid-inducible insulin-like growth factor-like peptide regulates adult development of the silkmoth Bombyx mori, FEBS J. 276: 1221-1232. PubMed ID: 19175674

Okamoto, N., et al. (2009b). A fat body-derived IGF-like peptide regulates postfeeding growth in Drosophila. Dev. Cell 17(6): 885-91. PubMed ID: 20059957

Pollak, M. (2008). Insulin and insulin-like growth factor signalling in neoplasia. Nat. Rev. Cancer 8: 915-928. PubMed ID: 19029956

Rusten, T. E., Lindmo, K., Juhasz, G., Sass, M., Seglen, P. O., Brech, A. and Stenmark, H. (2004). Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Dev. Cell 7: 179-192. PubMed ID: 15296715

Saltiel, A. R. and Kahn, C. R. (2001). Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414: 799-806. PubMed ID: 11742412

Scott, R. C., Schuldiner, O., and Neufeld, T. P. (2004). Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev. Cell 7: 167-178. PubMed ID: 15296714

Slaidina, M., et al. (2009). A Drosophila insulin-like peptide promotes growth during nonfeeding states. Dev. Cell 17(6): 874-84. PubMed ID: 20059956

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

Zhang, H., et al. (2009). Deletion of Drosophila insulin-like peptides causes growth defects and metabolic abnormalities. Proc. Natl. Acad. Sci. 106(46): 19617-22. PubMed ID: 19887630

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

date revised: 25 April 2024

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.