REVIEWS

How flies get their size: genetics

meets physiology
Bruce 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.

Division of Basic Sciences,
Fred Hutchinson Cancer
Research Center, 1100
Fairview Avenue North,
B2–152, Seattle, Washington
98109, USA.
e-mail: bedgar@fhcrc.org
doi:10.1038/nrg1989
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 development
Insects 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 later 5,6. 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

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Fat body viable weight is achieved delays the time course to pupa-
A mesoderm-derived energy- tion5, and so mimimum viable weight occurs a few hours
storage organ that fulfils the earlier and is less than critical weight. Nevertheless,
functions that are assumed by because minimum viable weight is simpler to measure
the liver and adipose tissues in
mammals.
experimentally, it has often been used interchangeably
with the term critical weight, especially in the Drosophila
Adipokinetic hormones literature10. Critical weight is determined primarily by
Peptide hormones with genotype, and is clearly affected by numerous loci10,11,
functions analogous to
as discussed below. In D. melanogaster, critical weight is
glucagons, produced by the
corpora cardiaca, a portion of
not significantly affected by diet4,6; slow-growing larvae,
the ring gland. These or larvae that are transiently starved, simply pass criti-
hormones stimulate the cal weight later. In M. sexta, however, poorer diets have
mobilization of stored fat and been found to decrease the critical weight8. It is unclear
carbohydrates from the fat
body upon starvation.
whether this difference is meaningful, or is due to dif-
ferences in the way critical weight has been measured in
Insulin-like peptides these two species.
(ILPs). Peptide hormones that It takes some time for an insect larva’s physiology to
are homologous to vertebrate
sense critical weight and then initiate the behaviours that
insulins and insulin-like growth
factors. ILPs are produced by
are associated with metamorphosis, including the cessa-
medial neurosecretory cells in tion of feeding. Larvae continue to grow during this lag
the brain, as well as the gut period, referred to as the ‘interval to cessation of growth’
and imaginal discs. These (ICG). Drosophila larvae grow so fast that they can more
ligands bind the insulin
receptor and promote cellular
than quadruple their weight during the ICG if they are
glucose import, energy storage cultured on rich food4. Therefore, although the normal
in the form of glycogen and dry weight for adult D. melanogaster in laboratory culture
triglycerides, and cell growth. is about 290 µg, adults as small as 35 µg can be obtained
Drosophila melanogaster has
by starving the larvae to prevent growth during the ICG4.
seven paralogous genes.
Orthologous genes in the silk
Growth trajectories during the ICG vary widely between
moth, Bombyx mori, are called species, and probably account for a significant amount
bombyxins. of species-specific size variation. M. sexta, for instance,
barely doubles its mass during the ICG, even when food
Cell autonomous
If the activity of a gene has
is not limiting 8. In this Review, I first outline what is
effects only in the cells that known about the control of growth rates, as the rate of
express it, its function is said to growth during the ICG is an important parameter that
be cell autonomous; if it causes controls final body size. I then address how achieving
effects in cells other than (or in
critical weight leads to the onset of metamorphosis,
addition to) those that express
it, its function is cell non-
which is the essence of how growth-phase duration is
autonomous. determined. Last, I delve into some unanswered ques-
tions related to allometry, or how the proportions of the
various organs are controlled.
Growth-rate control
Growth rates during larval development are affected by
nutrition, temperature (BOX 1), the density of animals in
their environment12 and, of course, genotype.
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.
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

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

ILPs Nutrition

Amino Glucose
acids
InR

PI3K AKT TSC1 ATP

a Starved
IRS TSC2 LKB1
PTEN AMPK
RHEB
b Fed Amino
acids
FOXO TOR

c Fed, ILP overexpression Transcription

S6K 4EBP TIF-IA
Growth Endocytosis
suppressors,
stress
response Translation Ribosomes Autophagy

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.

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

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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.
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 body 43,44. 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-
20-hydroxyecdysone ological differences that have been noted. Starvation of
(20E). The insect moulting
M. sexta larvae, for instance, rapidly arrests growth of the
hormone, a steroid. The
pro-hormone ecdysone is imaginal discs45 (BOX 3).
produced by the prothoracic Because dietary protein, and not sugar, is required
gland. Ecdysone is then for cell growth, one might expect the mNSCs, which
converted into active 20E produce the ILPs, to be sensors of dietary protein.
by the fat body, malpighian
tubules (analogous to the
However, experiments indicate that it is sugar, not
kidneys) and possibly protein, that mNSCs require to express ILP mRNA in
the epidermis. D. melanogaster 27,36. Consistently, injection of glucose
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into starved Bombyx mori (silk moth) larvae is sufficient
to stimulate the secretion of bombyxin 41. 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
www.nature.com/reviews/genetics

JH PTTH
ILP
JHE CA
PG mNSCs
Critical weight 20E ? ILP Nutrition
Fat body
?
Cessation of
feeding Peripheral
metamorphosis tissue growth
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.
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,
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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 control
Changes 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 pathway 62. 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
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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.
Box 4 | Hormonal controls that time metamorphosis
Weight
JH
20E
PTTH
ICG
1st moult 2nd moult Critical Cessation Pupariation
weight of growth
attained
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.
912 | DECEMBER 2006 | VOLUME 7
© 2006 Nature Publishing Group
Growth-phase duration
The 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-
tally 9,18 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
www.nature.com/reviews/genetics

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.
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
Juvenile hormone second confounding observation is that mutant larvae
(JH). A sesquiterpene produced that lack imaginal discs altogether can pupariate69,70,
by the corpora allata, which is a although this occurs later and at a smaller size than nor-
portion of the ring gland. JH
promotes juvenile (larval)
mal71. Therefore, although disc growth might provide a
development and inhibits ‘growth checkpoint’ in some insects, there must be an
metamorphosis. It is degraded, underlying disc-independent mechanism that can sense
in part, by juvenile hormone critical weight and trigger pupariation.
esterase, which is produced by
Despite these puzzling discrepancies, recent find-
the fat body and malpighian
tubules (kidney). ings36,51–53 support at least one potential size-sensing
mechanism that is consistent with most of the literature
Prothoracicotropic hormone and anecdotal accounts. If the imaginal discs, gut and
(PTTH). A neuropetide that is salivary glands produce ILPs27, the exponential growth
secreted by cells in the brain.
PTTH stimulates ecdysone
of these organs might cause an exponential increase
production by the prothoracic in IIS to the prothoracic gland, and thereby promote
gland. its increasing production of ecdysone. Because 20E
NATURE REVIEWS | GENETICS
© 2006 Nature Publishing Group
REVIEWS
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 allometry
So 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
VOLUME 7 | DECEMBER 2006 | 913
REVIEWS
PCG ICG
Passed Yes Yes
Last larval JH titres JH cleared from
critical
instar decline haemolymph?
weight?
No No
Continue Continue
growing growing
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.
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
914 | DECEMBER 2006 | VOLUME 7
© 2006 Nature Publishing Group
Yes Secrete PTTH Stop feeding
Photoperiodic
Stop growing
gate open? and ecdysone
Begin metamorphosis
No
Continue
growing
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 directions
The 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.
www.nature.com/reviews/genetics

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Competing interests statement
The author declares no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
AKT | chico | 4EBP | FOXO | HH | InR | Notch | PDK1 | PTEN |
S6K | TIF-IA | TOR | WG
UniProtKB: http://ca.expasy.org/sprot
Myc
FURTHER INFORMATION
Edgar laboratory homepage:
http://www.fhcrc.org/science/labs/edgar
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