How flies get their size: genetics meets physiology

How growth, size and form are controlled during animal development are problems that have entranced biologists for over a century. Recently, converging studies of cell growth and proliferation, pattern formation, endocrine regulation and evolution have generated new perspectives to these problems. Progress in the insect model systems, as reviewed here, has been particularly noteworthy, as genetic studies in Drosophila melanogaster have con-verged with classical endocrinological studies in other insects to generate a hypothesis to explain body size, if not shape and form. The physiology of growth control in insects is, of course, different to that in mammals, but the genes and signalling pathways that are involved are surprisingly similar. For instance, insulin/insulin-like growth factor signalling (IIS) controls rates of cell growth, nutrient use, cell size and body size in both flies and mice, and steroid hormone–nuclear receptor pairs regulate life-stage transitions that affect growth in both. Therefore, advances in insects, particularly in D. melanogaster, are influencing parallel studies in mice. Here I review recent findings that are relevant to the control of body size in D. melanogaster, with reference to other insects in the many cases in which it is enlightening.

Insect developmentInsects develop through a sequence of larval stages called instars, which are interrupted by moults in which the animal sheds its old exoskeleton and dons a new, larger one. Following a genetically specified number of moulting cycles, many insects enter a pupal stage, dur-ing which they undergo metamorphosis to their adult,

reproductive form. During metamorphosis, undifferenti-ated progenitor cells, most of which are termed imaginal cells, replace the older larval-specific cells, remodel the existing organs and generate new ones, such as wings and eyes, de novo. Completely metamorphosing insects (‘holometabolous’ insects) include common winged species such as flies (Diptera), butterflies and moths (Lepidoptera), ants and bees (Hymenoptera) and beetles (Coleoptera). Insects do not grow as adults, and so their final size can be considered, to a first approximation, as a product of their growth rate during the larval phases and the duration of this growth period1–3.

Critical weight and the ICG. One concept that is central to this discussion is target size, which varies from spe-cies to species and is therefore genetically determined. In holometabolous insects, the first manifestation of target size is the commitment to metamorphosis. This occurs sometime after the growing larva has passed a weight threshold that is termed the ‘minimum viable weight’, operationally defined as the weight at which larvae can develop into adults if food is completely withdrawn. This threshold was first recognized in D. melanogaster by Beadle4 in the 1930s, and was distin-guished from a more biologically relevant one, termed ‘critical weight’, some years later5,6. Critical weight has most often been defined as the weight after which feeding no longer affects the time course to pupation, and has been studied most carefully in Manduca sexta7–9, a large moth that is a favourite system for studies of insect physiology. In D. melanogaster, withdrawing food just as minimum

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, B2–152, Seattle, Washington 98109, USA.e-mail: [email protected]:10.1038/nrg1989

How flies get their size: genetics meets physiologyBruce A. Edgar

Abstract | Body size affects important fitness variables such as mate selection, predation and tolerance to heat, cold and starvation. It is therefore subject to intense evolutionary selection. Recent genetic and physiological studies in insects are providing predictions as to which gene systems are likely to be targeted in selecting for changes in body size. These studies highlight genes and pathways that also control size in mammals: insects use insulin-like growth factor (IGF) and Target of rapamycin (TOR) kinase signalling to coordinate nutrition with cell growth, and steroid and neuropeptide hormones to terminate feeding after a genetically encoded target weight is achieved. However, we still understand little about how size is actually sensed, or how organ-intrinsic size controls interface with whole-body physiology.

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© 2006 Nature Publishing Group

Fat bodyA mesoderm-derived energy-storage organ that fulfils the functions that are assumed by the liver and adipose tissues in mammals.

Adipokinetic hormonesPeptide hormones with functions analogous to glucagons, produced by the corpora cardiaca, a portion of the ring gland. These hormones stimulate the mobilization of stored fat and carbohydrates from the fat body upon starvation.

Insulin-like peptides (ILPs). Peptide hormones that are homologous to vertebrate insulins and insulin-like growth factors. ILPs are produced by medial neurosecretory cells in the brain, as well as the gut and imaginal discs. These ligands bind the insulin receptor and promote cellular glucose import, energy storage in the form of glycogen and triglycerides, and cell growth. Drosophila melanogaster has seven paralogous genes. Orthologous genes in the silk moth, Bombyx mori, are called bombyxins.

Cell autonomous If the activity of a gene has effects only in the cells that express it, its function is said to be cell autonomous; if it causes effects in cells other than (or in addition to) those that express it, its function is cell non-autonomous.

viable weight is achieved delays the time course to pupa-tion5, and so mimimum viable weight occurs a few hours earlier and is less than critical weight. Nevertheless, because minimum viable weight is simpler to measure experimentally, it has often been used interchangeably with the term critical weight, especially in the Drosophila literature10. Critical weight is determined primarily by genotype, and is clearly affected by numerous loci10,11,

as discussed below. In D. melanogaster, critical weight is not significantly affected by diet4,6; slow-growing larvae, or larvae that are transiently starved, simply pass criti-cal weight later. In M. sexta, however, poorer diets have been found to decrease the critical weight8. It is unclear whether this difference is meaningful, or is due to dif-ferences in the way critical weight has been measured in these two species.

It takes some time for an insect larva’s physiology to sense critical weight and then initiate the behaviours that are associated with metamorphosis, including the cessa-tion of feeding. Larvae continue to grow during this lag period, referred to as the ‘interval to cessation of growth’ (ICG). Drosophila larvae grow so fast that they can more than quadruple their weight during the ICG if they are cultured on rich food4. Therefore, although the normal dry weight for adult D. melanogaster in laboratory culture is about 290 µg, adults as small as 35 µg can be obtained by starving the larvae to prevent growth during the ICG4. Growth trajectories during the ICG vary widely between species, and probably account for a significant amount of species-specific size variation. M. sexta, for instance, barely doubles its mass during the ICG, even when food is not limiting 8. In this Review, I first outline what is known about the control of growth rates, as the rate of growth during the ICG is an important parameter that controls final body size. I then address how achieving critical weight leads to the onset of metamorphosis, which is the essence of how growth-phase duration is determined. Last, I delve into some unanswered ques-tions related to allometry, or how the proportions of the various organs are controlled.

Growth-rate controlGrowth rates during larval development are affected by nutrition, temperature (BOX 1), the density of animals in their environment12 and, of course, genotype.

Nutrition. Drosophila melanogaster larval development is complete after 4 days on rich food at 25oC, but can be extended to several weeks by restricting dietary pro-tein. Not surprisingly, larval growth can be arrested by removing dietary protein completely. This treatment rapidly arrests cell growth and DNA replication in most of the differentiated larval-specific tissues, but if the minimum viable weight has been attained, the progeni-tor cells that will form the adult continue to grow and proliferate13, eventually generating a small but otherwise normal fly. The fact that cells can grow within a starved animal indicates that the haemolymph (blood fluid) in such animals is not critically depleted of nutrients. Indeed, D. melanogaster and other insects are known to maintain haemolymph nutrients when they are starved by mobilizing triglycerides and glycogen stored in the fat body. Nutrient mobilization is mediated by the induction of metabolic neuropeptides called adipokinetic hormones (AKH), which are produced by the corpora cardiaca, a region of the neuroendocrine ring gland. AKHs function analogously to vertebrate glucagons14,15 and, together with insulin-like peptides (ILPs), are part of an endocrine signalling system that allows the animal to coordinate rates of cell growth and changes in diet, with minimal disruption of the developmental programme.

Insulin/insulin-like growth factor signalling. The insect IIS system (BOX 2) is highly homologous to that found in mammals. IIS activity promotes glucose import and nutrient storage by the fat body and other organs, fulfill-ing the homeostatic function of vertebrate insulins16–18. In this capacity, it affects feeding behaviour, lifespan and reproduction19. During development, IIS also regulates cell growth, fulfilling the developmental function of the mammalian insulin-like growth factors (IGFs)20,21. IIS activity has been manipulated in various ways in D. melanogaster, using the Gal4–UAS system to overex-press genes in specific tissues, and the Flp–FRT system to delete gene functions in specific tissues at defined times22. These manipulations show that many IIS components are not only essential for cell and organ growth, but are also sufficient to autonomously increase the growth rate of just about any cell type in D. melanogaster 23–26 (BOX 2). In whole animals, increased expression of several of D. melanogaster’s seven ILPs can increase both larval growth rates and adult size17,27,28, whereas ablation of the small cluster of medial neurosecretory cells (mNSC) in the brain, which are the principal source of ILPs, reduces growth rates and final body size16,17,28. Studies using a temperature-sensitive allele of the insulin receptor (InR)29 to regulate IIS at defined stages showed that the role of IIS as a regulator of growth rate is general, but that its effects on body size are limited to the ICG30. This supports earlier studies, in which changes in diet were used to show that the commitment to metamorphosis depends on achieving a critical size, rather than time or growth rate.

The Target of rapamycin (TOR) protein kinase is the best characterized, and arguably, the most important, growth-regulatory target of insulin signalling23,31,32, at least in well-fed animals33,34. Studies in cultured cells show that, in addition to sensing nutritional state indirectly

Box 1 | Temperature and body size

Body size in ectotherms is generally affected by temperature80. In insects, lower temperatures decrease growth rates but actually increase final body size81. This affect has been attributed to increased cell sizes rather than altered cell numbers82. Davidowitz and Nijhout79 showed that, in Manduca sexta, growth rates increase linearly with temperature, and proposed a simple explanation of why body and cell size nevertheless decrease. They show that the interval to cessation of growth (ICG) decreases markedly with increasing temperature, and that this more than counteracts the effect of faster growth rates. So, at lower temperatures, the longer ICG is presumed to allow more net growth, even though the growth rate is slower. This simple explanation fits the experimental data well79, but leaves open the question of why the ICG is selectively affected at higher temperatures. This presumably derives from the differential effects of temperature on feeding and growth, on the one hand, and the kinetics of hormonal fluxes, on the other.

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PI3K

PTEN

AKT

FOXO

LKB1

InR

TSC2

TOR

TSC1

Aminoacids

TIF-IA4EBPS6K

AMPK

ATP

Aminoacids

Glucose

IRS

Endocytosis

Transcription

AutophagyRibosomes

Growth suppressors,stress response Translation

ILPs Nutrition

RHEB

a Starved

c Fed, ILP overexpression

b Fed

Intracellular second messenger A signalling molecule in cells, the concentration of which changes on the binding of an extracellular ligand to a plasma-membrane-bound receptor.

through IIS, TOR responds cell autonomously to levels of cellular amino acids, ATP and oxygen35. Although it is doubtful that cells inside an insect larva ever experience absolute amino-acid or glucose depletion as practised in cell-culture experiments, experiments in D. melanogaster indicate that certain larval cells that are resident in the fat body and perhaps other endocrine organs might use TOR to sense the smaller fluctuations in haemolymph nutrients that accompany changes in diet36,37.

There is a finely tuned, elaborate feedback network between IIS, TOR and their nutritional inputs. Glucose import, which is controlled by IIS, probably affects cel-lular ATP pools, which control TOR activity. TOR also feeds back on IIS in at least two ways. First, TOR nega-tively regulates ISS through feedback between the kinase S6K and insulin receptor substrate (IRS) proteins37, and, second, TOR activity is required to activate AKT,

a crucial IIS transduction component38 (BOX 2). The first mechanism might dampen the cellular response to ILP signalling when nutrient levels are high, whereas the second mechanism presumably blocks the potentially harmful effects of inappropriate IIS when cells are criti-cally starved for amino acids, glucose or oxygen. Another homeostatic mechanism involves the transcription fac-tor FOXO, which activates transcription of the InR, but is also repressed by IIS activity through AKT39.

Numerous observations underscore the importance of IIS and TOR signalling as a nutrient-response sys-tem. Starvation represses the expression of several of the ILPs27,28,36, depletes the IIS second messenger PIP3 (REF. 24), and inhibits the activity of the TOR target S6K (REF. 40). Consistent with this, a silk moth ILP, bombyxin, is depleted from the haemolymph on starvation41. Perhaps most significant of all, genetically activating IIS

Box 2 | The insulin/insulin-like growth factor signalling system

The Drosophila melanogaster insulin/insulin-like growth factor signalling (IIS) system has been the subject of intense genetic analysis, and is essentially similar to its human counterpart. It comprises a group of seven insulin-like peptides (ILPs), a single insulin receptor (InR) gene, an insulin receptor substrate (IRS) protein called chico, the type IA phosphati-dylinositol 3-kinase (PI3K), the lipid phosphatase PTEN and the protein kinases AKT/PKB and PDK1 (REF. 23). InR and its downstream effectors are expressed ubiquitously, but the seven ILPs are expressed in specific tissues, presumably in response to different inputs27,28. These ligands are nevertheless thought to mediate common cellular effects through the same receptor. IIS affects growth by promoting the cellular import of glucose to enhance the cell’s energy supply, by inhibiting the transcriptional activator FOXO33,83, which activates metabolic repressors such as 4EBP, and by maintaining the activity of the Target of rapamycin (TOR), a conserved protein kinase. TOR is activated by the small GTPase RHEB and promotes translational initiation, ribosome biogenesis, nutrient storage and endocytosis, and inhibits autophagy35,84. In addition to regulation by IIS, TOR activity is also sensitive to cellular levels of amino acids and the ATP:ADP ratio, which is an index of cellular energy levels. TOR senses energy levels through AMP-dependent kinase (AMPK), which is activated by the LKB1 protein kinase, and inhibits the TSC1–TSC2 complex. It remains unclear how TOR senses amino-acid levels. TIF-IA is a transcription factor that stimulates rRNA synthesis.

In the diagram, IIS factors are in blue, TOR-pathway components are shown in red and AMPK-pathway components are shown in green.

The inset shows male flies, from the top down: part a, subjected to dietary restriction for protein; part b, raised on a rich diet; and part c, subjected to constitutive, systemic ILP overexpression, also raised on rich food. Photographs courtesy of S. Layalle, C. Géminard and P. Leopold, University of Nice.

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20-hydroxyecdysone (20E). The insect moulting hormone, a steroid. The pro-hormone ecdysone is produced by the prothoracic gland. Ecdysone is then converted into active 20E by the fat body, malpighian tubules (analogous to the kidneys) and possibly the epidermis.

or TOR signalling can bypass the known cellular effects of starvation, including the arrest of cell growth and DNA replication in the larval-specific tissues24,42, and the induction of autophagy in the fat body43,44. These obser-vations indicate that IIS and TOR activity are suppressed in the larval-specific tissues by starvation, and that this systemically mediates a set of cellular responses that allow insect larvae to cope with fluctuating nutritional conditions.

As noted above, adult progenitor cells in the brain and imaginal discs are relatively insensitive to changes in diet. In D. melanogaster, growth and division of these cells is not arrested in animals that are starved after reaching critical weight. These cells might require less of the ILP stimulus to maintain high rates of anabolic metabolism, or they might produce their own ILPs. Indeed, starvation-insensitive ILP expression has been documented in both locations27,28. The nutrition-independent growth of progenitor cells in D. melanogaster larvae probably reflects the animal’s life strategy, which is to prioritize nutrient utilization for generating reproduc-tively capable adults. Of course, this strategy succeeds only if an animal has achieved minimum viable weight before it is starved. Animals that are starved before this do not have sufficient stored nutrients to support growth of the imaginal cells to term, and they eventually perish. In insects other than D. melanogaster, the developmen-tal strategy for achieving the same life strategy can be different, and this is reflected in some of the physi-ological differences that have been noted. Starvation of M. sexta larvae, for instance, rapidly arrests growth of the imaginal discs45 (BOX 3).

Because dietary protein, and not sugar, is required for cell growth, one might expect the mNSCs, which produce the ILPs, to be sensors of dietary protein. However, experiments indicate that it is sugar, not protein, that mNSCs require to express ILP mRNA in D. melanogaster 27,36. Consistently, injection of glucose

into starved Bombyx mori (silk moth) larvae is sufficient to stimulate the secretion of bombyxin41. Therefore, although it is clear that the IIS system somehow senses dietary protein, the underlying mechanism remains obscure. It has been suggested that ILP secretion from the mNSCs might be regulated by dietary protein, but it seems equally likely that some other organ senses amino acids and promotes IIS activity indirectly, through secondary factors.

The fat body. This other nutrient-sensing organ could be the fat body. Although the fat body has not been reported to produce ILPs27,28, organ-culture experi-ments indicate that it does produce some sort of growth factor13,46, and that this might be nutrient dependent. Recent reports show that specifically suppressing metabolism in the fat body by inhibiting amino-acid import, genetically squelching TOR activity or activat-ing FOXO is sufficient to non-autonomously suppress IIS activity in both larvae36 and adults47. The nutrition-dependent production of an IIS cofactor by the fat body is one attractive explanation for these results (FIG. 1). The role of TOR in the fat body might be either to sense amino-acid and ATP pools, or to promote the synthe-sis of this putative IIS cofactor, or both. A putative insulin-binding factor, acid labile subunit (ALS), which is produced by the fat body only in feeding animals36, is a candidate for this missing growth factor. Other can-didates are the chitinase-related imaginal disc growth factors (IDGFs)48 and adenosine-deaminase-related growth factor D (ADGFD)49,50, which are mitogens that are expressed by the fat body.

Ecdysone and the prothoracic gland. Virtually all bio-logical regulatory systems have elements of negative feedback that keep them from running wild. An impor-tant mode of negative feedback in body-size control seems to involve 20-hydroxyecdysone (20E)51–53, the ster-oid hormone that controls both moulting and the onset of metamorphosis in all insects54,55. Recent studies found that 20E activity is positively controlled by IIS activity in the prothoracic gland, a sector of the ring gland that produces the steroid hormone ecdysone, which is the 20E precursor51–53. Suppressing IIS in the prothoracic gland reduces 20E activity and increases adult body size, whereas increasing IIS has the opposite effect. How IIS stimulates ecdysone production is still unclear, but the same relationship has been documented in other organs in mosquitos and silk moths19, and therefore is probably general. Other factors that affect cell growth, such as Myc and cyclin D–CDK4, do not seem to stimulate ecdysone production by the prothoracic gland, whereas activating the Ras–Raf pathway does53. The explanation for this is not clear, but one possibility is that both IIS and Ras–Raf signalling promote specific aspects of metabolism that are required for steroid synthesis.

It was expected that the body-size effects that result from altering the prothoracic gland would be due to changes in the timing of the onset of metamorphosis, which is triggered when the prothoracic gland releases a large pulse of ecdysone (BOX 4). Indeed, extreme changes

Box 3 | Another function for juvenile hormone

Juvenile hormone (JH) has been well characterized as a suppressor of prothoracicotropic hormone (PTTH) and 20-hydroxyecdysone (20E) release, but in Manduca sexta it also interacts with the insulin/insulin-like growth factor (IIS) system to suppress the formation and growth of late-forming imaginal discs45. Such discs, unlike the extensively studied early-forming wing and eye discs of Drosophila melanogaster, undergo most of their growth after the onset of metamorphosis and pupariation, using nutrients stored in earlier stages. Drosophila melanogaster has analogous sets of imaginal cells: examples include the nests of histoblasts that generate the adult abdominal epidermis and the islands of imaginal cells that reside in the larval gut. The formation and growth of the late-forming eye and leg discs in M. sexta are arrested by starvation before attainment of critical weight, but they will form and grow in starved animals from which the corpora allata, the JH-producing organ, has been excised45. These effects are ecdysone independent, suggesting that JH might function directly as a negative growth factor, at least on eye, leg and wing imaginal cells. However, the in vivo application of a stable JH mimetic does not suppress the formation or growth of the late discs85, indicating that the growth-suppressive action of JH is eventually overcome by nutrient-dependent signals (for example, IIS) in feeding animals. The molecular targets by which JH suppresses imaginal cell growth remain unknown, as does the general mode of JH signal transduction86. Whether JH modulates growth in tissues other than the late-forming discs or in insects other than M. sexta are also open questions.

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mNSCsPG

CA

20E

Fat body

?

?

JH PTTH

Peripheraltissue growth

Cessation offeedingmetamorphosis

NutritionCritical weight

JHE

ILP

ILP

in IIS activity in the prothoracic gland do change the timing of metamorphosis, and therefore probably also alter the duration of the ICG52,53. Surprisingly, how-ever, Colombani51 and Mirth52 found that more subtle changes in IIS in the prothoracic gland clearly affected rates of larval growth from early development, with-out affecting developmental timing. Closer inspection revealed that 20E and its receptor, EcR, antagonize the ability of ILP signalling to activate PI3K and AKT, and to suppress the nuclear localization of FOXO51 (BOX 2). The effect on FOXO was found to be cell autonomous, at least in the fat body, implying that IIS and 20E proba-bly have antagonistic effects on growth and metabolism at the cellular level. Consistent with the role of the fat body in whole-body growth control, ecdysone signal-ling to the fat body has non-cell-autonomous, systemic effects on growth. For instance, specifically suppressing ecdysone signalling to the fat body increases overall growth rates and final body size, without affecting development time. An attractive explanation for this is that ecdysone signalling suppresses the production of nutrient-dependent growth factors by the fat body (FIG. 1). However, the role of 20E as a growth factor is far from simple; observations in other insects show that,

although high 20E levels inhibit cell proliferation, low levels of 20E or ecdysone are required for the growth and proliferation of some larval cells in culture56,57. The ability of ecdysone to antagonize IIS is therefore either level dependent, or perhaps cell-type specific. Further studies addressing the interface of ecdysone, IIS and TOR signalling are required to clarify this interesting and unexpected connection.

Cell-size controlChanges in cell size account for a significant amount of the variation in body size that is seen in D. melanogaster, both in wild populations58 and experimental situa-tions32,33. The size of proliferating cells is determined by their relative rates of growth and division. I have discussed growth-rate control, but how are division rates regulated? Rates of cell division in the imaginal discs are clearly growth dependent: when growth rates are depressed by lack of nutrition, IIS or TOR signalling, cell division slows down too. However, experimental manipulations that upregulate cell growth rates generally do not increase the speed or number of cell divisions in these organs23,59, at least when the experiments are performed on rich food. Therefore, there seems to be an intrinsic developmental programme that determines the maximal rate of division of the imaginal cells. As discussed below, the final number of cells in each organ is also developmentally programmed, largely inde-pendently of growth rate. It is partly because of these independent controls on cell division rates and cell numbers that nutrition, IIS and TOR signalling have such limited effects on final cell numbers in the adult but such profound effects on cell size: if cell growth rates are increased without coincidentally increasing division, cell size increases.

Adult insects consist almost completely of non-proliferating cells, and so the sizes of their cells are determined simply by the relative rates of synthesis, storage and turnover of macromolecules, metabolites and water. Accordingly, most of the genes that affect cell size in adults alter metabolic balance in some way. This includes the IIS- and TOR-signalling genes that were discussed above (BOX 2), and a few others such as Myc and the cyclin D–CDK4 kinase59–61, which affect post-mitotic cell growth. Like TOR, Myc is a potent stimulator of ribosome biogenesis and protein synthesis61, but seems to be regulated by IIS- and TOR-independent, non-nutrition-related developmental signals. Cyclin D–CDK4 is a non-essential regulator of cell growth that has a separate function in controlling cell-cycle progression through the conserved E2F–RB pathway62. Cyclin D–CDK4 upregulates mitochondrial activity by unknown mechanisms, and requires intact mitochondrial function to stimulate cell growth60. Genetic tests show that each of these gene functions affects not only cell size, but body size as well, and therefore they are potential targets for evolutionary selection of body size.

Population-genetics studies have also documented significant differences in cell size between large and small strains of D. melanogaster 58,63, although the genetic basis of these changes has only been guessed

Figure 1 | Endocrine communication between the organs involved in growth control. Organs and cells are denoted as ovals, hormones are denoted as coloured boxes and effects are denoted in grey boxes. Nutrition allows growth of tissues throughout the body, and also stimulates the production of insulin-like peptides (ILPs) by medial neurosecretory cells (mNSCs) in the brain. ILPs activate the insulin/insulin like growth factor–Target of rapamycin (IIS–TOR) system and promote growth systemically. The fat body also senses nutrition, probably using TOR, and produces poorly characterized growth factors that might potentiate IIS. IIS to the prothoracic gland (PG) promotes the production of 20-hydroxyecdysone (20E) by this organ, providing negative feedback that supresses cell growth and, eventually, the cessation of feeding and the onset of metamorphosis. Juvenile hormone (JH), juvenile hormone esterase (JHE) and prothoracicotropic hormone (PTTH) provide another negative-feedback loop. JH is produced by the corpora allata (CA) in young growing animals, and suppresses 20E levels by inhibiting PTTH release. However, when the growing animal achieves critical weight, rising levels of JHE (produced by the fat body and other organs) degrade JH, leading to the release of PTTH, which triggers ecdysone production. This in turn suppresses growth, ends feeding and causes metamorphosis.

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ICG1st moult 2nd moult Critical

weight attained

PupariationCessation of growth

WeightJH20EPTTH

at (BOX 5). It is logical to assume that adult cell sizes, and even organ sizes, might be determined in part by differentially programmed expression of IIS- and TOR-pathway components58,64, and at least one recent study supports this view with data18. However, virtu-ally any factor that alters the balance between anabolic and catabolic metabolism could be expected to act as a determinant of cell size.

Growth-phase durationThe timing of the cessation of feeding and the onset of metamorphosis is a critical determinant of body size in insects. This transition is controlled, at least in part, by a physiological network of hormones that circulate in the animal’s haemolymph54 (FIG. 1). As outlined in BOX 4, the growth phase is terminated by a hormonal cas-cade, which starts with the attainment of critical weight and ends with the secretion of high levels of ecdysone, triggering the cessation of feeding and pupariation. In M. sexta, the signal that critical weight has been reached is relayed by a drop in juvenile hormone (JH), followed by secretion of prothoracicotropic hormone (PTTH), which triggers ecdysone production (FIG. 1). The same is assumed to be true in D. melanogaster, although comprehensive data are lacking.

Models for size control. Davidowitz et al.8, Nijhout et al.9

and Shingleton et al.18 have elaborated on the early find-ings of Beadle4, Bakker5 and others to develop models for the physiological control of body size. These models describe in specific terms the interplay between growth rate, critical weight and the control of JH, PTTH and ecdysone during the final larval instar of D. melanogaster or M. sexta development. Both have three critical param-eters: the growth rate, the critical weight and the length of the ICG (FIG. 2). After critical weight is attained, the animal continues to grow until a spike of 20E causes the cessation of feeding and the onset of pupariation and metamorphosis (BOX 4). Because larval growth is essentially exponential, growth during the ICG can be substantial, accounting for about half of the peak larval mass in M. sexta and most of it in D. melanogaster, pro-vided that growth occurs on rich food. If growth is slowed or stopped during the ICG by poor diet, loss of insulin sig-nalling or crowding, for instance, then the final weight of the animal will be much closer to its critical weight. This model has been carefully tested both experimen-tally9,18 and by computer-based simulations9, and seems to be a useful framework for understanding the effects of nutrition, IIS and ecdysone signalling on body-size control in Diptera and Lepidoptera. It provides a simple explanation for the effects of diet and IIS , because these factors have big effects on growth rates during the ICG but relatively insignificant effects on critical weight4,8,18. However, the current paradigm has limitations. One is that it applies only to the last instar of larval develop-ment, and leaves open the important question of how critical weight is determined and sensed, and how this information is then relayed to the ring gland to end JH production and induce PTTH and ecdysone production. This question would seem to be at the heart of overall size control, at least in Diptera and Lepidoptera.

Sensing critical weight. So far, little is known about what parameter is actually ‘read’ as critical weight. However, there are some thought-provoking observations to con-sider. For instance, early studies in several insects showed that the presence of growing or regenerating discs delays pupariation65–67, indicating that growing imaginal tissues generate a signal that maintains JH at high levels until

Box 4 | Hormonal controls that time metamorphosis

Classic work in several insects showed that the cessation of feeding and onset of metamorphosis are triggered by pulses of circulating 20-hydroxyecdysone (20E), the same steroid hormone that triggers earlier moults. The graph is an idealized schematic that represents hormone fluxes in the haemolymph of a growing larva. Attainment of critical weight suppresses juvenile hormone (JH) production, leading to prothoracicotropic hormone (PTTH) production, ecdysone secretion and the cessation of feeding. A large fraction of total growth occurs in the interval to cessation of growth (ICG).

Insects produce ecdysone in their prothoracic glands, which sense multiple inputs. Insulin/insulin-like growth factor (IIS) and Ras–Raf signalling to the prothoracic gland affect 20E levels51–53, suggesting that 20E production is controlled by nutritional and cell-specification signals, respectively. In Bombyx mori, 20E production can also be controlled by direct innervation of the prothoracic gland87, presumably from the brain. However, the pulses of 20E that trigger moulting, the cessation of feeding and pupariation are triggered by PTTH, which is secreted by cells in the nearby brain. In some insects, such as milkweed bugs (Oncopeltus fasciatus) and blood-sucking Hemitpera (Rodnius prolixus and Dipetalogaster maximus), PTTH secretion is triggered by a size-measuring mechanism that involves stretch receptors in the abdomen. When the abdomen is distended by feeding, these stretch receptors signal to the brain to release the hormone2. However, this mechanism is not universal. In Onthophagus taurus, a beetle, PTTH secretion is triggered when the larva’s food supply is exhausted88, and in Manduca sexta, a moth, PTTH secretion is inhibited by JH, a sesquiterpene hormone that is present through early development and then cleared from the haemolymph when the larva reaches a critical weight86. The loss of JH is due, in part, to a rise in the activity of juvenile hormone esterase (JHE), an enzyme that breaks down JH and is produced by the fat body in feeding animals72. In M. sexta, JH levels fail to drop if larvae are starved before attaining critical weight, and experimental application of JH can block pupation and extend the period of larval growth, giving rise to oversize larvae. In Drosophila melanogaster, excess JH also prolongs the final (third) instar, but this treatment does not lead to larger animals89. PTTH secretion in M. sexta, is further regulated by a photoperiodic ‘gate’90, such that it can only be released during an 8-hour window each day. Therefore, the time of day at which the larva clears its JH is another variable that affects body size (FIG. 2). This control has not been demonstrated in D. melanogaster, although recent work hints at its existence52. Figure after REFS 54,91.

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Juvenile hormone (JH). A sesquiterpene produced by the corpora allata, which is a portion of the ring gland. JH promotes juvenile (larval) development and inhibits metamorphosis. It is degraded, in part, by juvenile hormone esterase, which is produced by the fat body and malpighian tubules (kidney).

Prothoracicotropic hormone(PTTH). A neuropetide that is secreted by cells in the brain. PTTH stimulates ecdysone production by the prothoracic gland.

their growth reaches some milestone, which could be a metric for critical weight. Consistent with this notion, mutations that disrupt cytoarchitecture in the imaginal disc cells, thereby allowing them to grow indefinitely, suppress 20E levels, prolong larval growth and, in some cases, generate oversize pupae65,67,68. But, although the idea that signals that originate from the discs act on the endocrine signalling centres to time metamorphosis has existed for many years, such signals have never been identified. Moreover, several facets of larval biology are hard to reconcile with this idea. For instance, the vari-ous imaginal tissues grow and mature asynchronously, and many continue to grow long after critical weight is achieved and even after the cessation of feeding. In insects other than flies, post-pupation disc growth is, in fact, the norm (BOX 3). These observations indicate that disc growth per se does not inhibit metamorphosis. A second confounding observation is that mutant larvae that lack imaginal discs altogether can pupariate69,70, although this occurs later and at a smaller size than nor-mal71. Therefore, although disc growth might provide a ‘growth checkpoint’ in some insects, there must be an underlying disc-independent mechanism that can sense critical weight and trigger pupariation.

Despite these puzzling discrepancies, recent find-ings36,51–53 support at least one potential size-sensing mechanism that is consistent with most of the literature and anecdotal accounts. If the imaginal discs, gut and salivary glands produce ILPs27, the exponential growth of these organs might cause an exponential increase in IIS to the prothoracic gland, and thereby promote its increasing production of ecdysone. Because 20E

suppresses cell growth51–53, rising levels would be expected to first slow growth in peripheral tissues, and then trigger the cessation of feeding. A second analogous feedback mechanism can be envisioned for the clearing of JH. In this case, exponential growth of the fat body, and a concurrent exponential rise in the capacity of this organ to produce juvenile hormone esterase (JHE)72, might help to clear JH and trigger the cascade that leads from PTTH to the production of 20E at a critical size. Similar feedback loops can be envisioned in which fat-body growth enhances 20E signalling through its ability to con-vert the ecdysone prohormone to active 20E, or through the production of growth factors that augment IIS activ-ity in the prothoracic gland36,48,50. Consistent with these ideas, increased IIS activity in the fat body causes not only increased fat-body growth, but precocious cessation of feeding24. The combined function of these sorts of negative-feedback loops might explain, in part, why larvae do not grow past critical size. However, these mechanisms have not been directly tested in the laboratory. How much of the haemolymph ILP is contributed by the discs and other tissues is unknown, studies of the fat body as a signalling centre have just begun, and the few studies of pupariation timing that have been published73 do not directly address these ideas. A comprehensive genetic analysis of the mechanisms that sense critical weight and control pupariation timing in D. melanogaster should add significantly to the already rich physiological literature from M. sexta, B. mori and other large insects.

Another limitation of the current paradigm (FIG. 2) is its assumption that adult size is proportional to peak larval size. Although this condition is consistent with much of the data from D. melanogaster and M. sexta, the available data also indicate that the maximal sizes of the adult organs are ultimately controlled by organ-intrinsic signalling, rather than as a function of total body size or stored nutrients (see below). If this is true, there is probably an upper limit on adult body size that is geneti-cally encoded, and that cannot be exceeded by simply increasing growth and nutrient storage during the ICG. It will be interesting to see whether increasing nutrient storage by larvae to three or four times the normal level results in proportional increases in adult size, or whether nutrients that are stored in excess of what is required to achieve a maximum, genetically programmed target size are simply discarded. Given the current state of the field, this should soon be possible in D. melanogaster.

Cell numbers and allometrySo far, experimental manipulations of nutrition and hormonal signalling have succeeded in reducing the weight of D. melanogaster adults to about 15% (REF. 4), or increasing it to about 150% (REF. 28), of what is normally attained in the laboratory when the flies are fed on rich food. Much of this variation is due to changes in cell size, rather than cell numbers, which in D. melanogaster have been decreased by genetic manipulation to about 80% (REFS 26,33), or increased to about 120% of nor-mal28,51. However, natural variation in cell numbers between species of Drosophila, or even subpopulations of D. melanogaster, are greater than this58,63,74. If one considers

Box 5 | Evolution of Drosophila melanogaster body size

Latitudinal clines in body size have been documented between Africa and Europe, from the Baltic to Central Asia, along the east coast of North America, and in Australia and Southern Africa. Flies with larger adult body size, faster development rate and higher fecundity reside at higher latitudes, corresponding to climates with cooler temperatures and harsher winters58. Conversely, smaller body size, slower development and decreased fecundity, but increased starvation resistance and longevity, are found in more equatorial populations. These adaptations are thought to reflect the different selective pressures in tropical versus temperate regions; fast development and thermotolerance are essential when flies must overwinter and then develop and breed quickly in the spring, whereas longevity and the ability to survive crowding and starvation are advantageous in warm climes. The main determinant of body-size variation in natural populations seems to be altered cell numbers (at least in the wings where cell numbers are most often tallied), but changes in cell size are also common63,92. Increased metabolic rate and efficiency have also been correlated with larger body size58.

The evolution of larger body size has been reproduced in several laboratories93,94, and most of the parameters that are seen to change in the wild, such as cell size, development rate and critical weight were found to be affected in a similar way. When small and large varieties of D. melanogaster are reared in identical conditions in the laboratory, their size differences and differences in critical weight breed true10,94, and are therefore genetically encoded. Several attempts have been made to genetically map QTLs that affect body size58,95, but these have so far failed to define the specific genes involved. Various authors have suggested that components of the insulin/insulin like growth factor–Target of rapamycin (IIS–TOR) signalling system could be important targets for selection of body size58,64, and most of the available data are consistent with this. Other factors, such as those that determine sensing of critical weight, the kinetics of juvenile hormone (JH) decline and prothoracicotropic hormone (PTTH) release, and the myriad determinants of the interval to cessation of growth (ICG) are also probable targets of selection.

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Stop feedingStop growingBegin metamorphosis

Photoperiodicgate open?

Secrete PTTHand ecdysone

Last larvalinstar

JH titresdecline

Passedcriticalweight?

JH cleared fromhaemolymph?

Continuegrowing

Continuegrowing

Continuegrowing

No No No

Yes Yes Yes

PCG ICG

more divergent species such as D. melanogaster and M. sexta, or mice and elephants, differences in cell numbers are vastly greater than differences in cell size. Therefore, from an evolutionary standpoint, the control of cell numbers is a more important determinant of body size than the control of growth rates or cell size. The control of cell numbers remains something of a mystery, and one that will probably not be resolved by studies of nutrition, IIS/TOR signalling and endocrinology. There are some clues, however.

One telling observation is that when imaginal discs are cultured for long periods in growth-permissive environments, such as young larvae or the abdomens of adults, they grow only to their normal target size and then stop75. Therefore, target cell number in these adult organs is essentially an organ-autonomous property. The same can be assumed of the CNS, which has a virtually invariant pattern of cell divisions13,76. The prevailing explanation for this is that organ size (and therefore, final cell numbers in the adult) are determined primarily by the way short-range, organ-autonomous (paracrine) signalling systems, such as the Wnt, bone morphogenic protein (BMP), hedgehog (HH), epidermal growth factor (EGF) and Notch pathways, are deployed dur-ing development. The specifics of local cell signalling, combined with organ-specific codes of transcriptional regulators (for example, Hox genes) seem to give each organ a genetically predetermined target size, which can be modulated over only a modest range by nutritional and endocrine inputs. Many genetic experiments with short-range signals and selector-type transcription fac-tors support this view (see REFS 1,3,59 for reviews). To take an example from mammals, the evolution of wings in bats is thought to derive from increased proliferation of the chondrocytes that lay down the finger bones. This has made the bat’s fingers, which form the wing, many times longer than the fingers of their mouse-like ances-tors. Increased chondrocyte proliferation in bat forelimb digits seems to derive from prolonged expression of a BMP, which functions as a local growth factor77.

In flies, short-range BMP signalling controls the tar-get size and shape of most of the organs discussed here, including imaginal discs, the ring gland, the fat body and various regions of the brain. Other local signals such as wingless (WG, a Wnt), HH, Notch, and the various ligands and receptors that feed into the Ras–MAPK pathway also

affect target size. Such short-range signals are commonly thought of as regulators of allometry, but considering how much communication there is between the various organs (FIG. 1), it would be surprising if short-range signals did not feed back at multiple levels on the endocrine signals that affect critical-weight sensing, growth rates and the duration of the growth period. Studies in beetles that document growth-regulatory interactions between differ-ent imaginal discs are consistent with such feedback64,78, and the discovery that Ras–MAPK signalling in the prothoracic gland affects 20E levels, pupariation timing and body size53 provides a tangible molecular example. Therefore, in considering the evolution of body size, there are few things that can be ignored.

Future directionsThe work discussed here is an inspiring example of how the convergence of two previously separate fields of study — Dipteran genetics and Lepidopteran physi-ology — can yield a new paradigm for understanding a sizeable biological issue. However, the synthesis is incomplete, because although most of the crucial organs and gene systems that control body size might have been identified, the way these units communicate (FIG. 1) is still poorly understood. Moreover, the size-control paradigm as it stands (FIG. 2) refers to a rather narrow window of time — the last instar of larval development — and aims to explain only the three- to fourfold variations in size that have been achieved experimentally, and that are seen within species in the wild. Understanding the much larger variations in body size between species that occur dur-ing evolution will require new concepts and approaches. Future studies focused on the functional connections between intrinsic size control in the imaginal tissues and critical-weight sensing by the whole animal are clearly important. It would be best if these studies were carried out using combined genetic and physiological tools in the same organism, to avoid the pitfalls of comparing systems (for example, D. melanogaster, M. sexta and B. mori) that have significant physiological differences. Incorporating evolutionary genetics is also an important goal, but this will be challenging, because comparative genomics without experimentation is unlikely to explain the intricacies of anatomy and physiology that, to a large extent, control how an organism regulates its growth rate and determines its final size.

Figure 2 | Control of growth during the final larval instar. The figure shows the checkpoint that governs the interval to cessation of growth (ICG), and the two checkpoints within the ICG before the cessation of growth. JH, juvenile hormone; PCG, growth prior to critical weight; PTTH, prothoracicotropic hormone. Figure modified from REF. 79. See also REF. 9.

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Competing interests statementThe author declares no competing financial interests.

DATABASESThe following terms in this article are linked online to:Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneAKT | chico | 4EBP | FOXO | HH | InR | Notch | PDK1 | PTEN | S6K | TIF-IA | TOR | WGUniProtKB: http://ca.expasy.org/sprotMyc

FURTHER INFORMATIONEdgar laboratory homepage: http://www.fhcrc.org/science/labs/edgarAccess to this links box is available online.

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How flies get their size: genetics meets physiology

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