Am J Physiol Regul Integr Comp Physiol 304: R177–R188, 2013.
First published December 5, 2012; doi:10.1152/ajpregu.00084.2012.
Review
Comparative approaches to the study of physiology: Drosophila as a
physiological tool
Wendi S. Neckameyer and Kathryn J. Argue
Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, St. Louis, Missouri
Submitted 22 February 2012; accepted in final form 5 December 2012
dorsal vessel; mechanosensation; mushroom bodies; transient receptor potential
channels; transgenics
THE EXTENSIVE TRACTABILITY of the Drosophila model system
has facilitated the discovery of numerous genes involved in
multiple physiological pathways that share tremendous conservation with mammalian systems. Although a fly is not a
human, there is no question that the basic pathways in cell,
tissue, and organ development and function are absolutely
conserved, and thus, information gleaned from understanding
fly physiology has had an enormous impact on our understanding of human physiology. In fact, without the “lowly” fruit fly,
our understanding would be greatly lacking for such critical
processes as programmed cell death, cardiac development, ion
channel function, and embryonic development, to name only a
very few research areas. What are the advantages to this
invertebrate model system that have enabled researchers to
elucidate components of so many different pathways?
Certainly, their rapid generation time (⬃10 days) is key (Fig. 1A).
The adult fly, upon emergence from the pupal case, is sexually
immature, and sexually mature behaviors, pheromonal complement, and gonadal development occur within 48 –72 h.
Senescence can commence within 2 wk, since a simple habituation paradigm is compromised by that time (88). Compare
this to months for rodents and years for humans.
Another major plus, especially during current times of significantly reduced funding, is their relative lack of expense to
maintain. On average, flies cost 20 cents/mo per 100 animals;
zebrafish are 900 times more expensive, and mice over 10,000
times the cost (66) (Fig. 1B).
But by far and away its arguably greatest advantage is the
incredible genetic tractability of Drosophila. Several crucial
Address for reprint requests and other correspondence: W. S Neckameyer, Dept.
of Pharmacological and Physiological Science, St. Louis Univ. School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104 (e-mail: [email protected]).
http://www.ajpregu.org
characteristics facilitated initial genetic studies in this animal.
There are only four chromosomes in the haploid Drosophila
genome, and numerous easily scored outward characteristics
have been identified that are also highly mutable. While the
contributions to classical genetics from early studies on Drosophila have been overwhelming, the range and complexity of
genetic and molecular tools now available have enabled Drosophila researchers to perform unbiased genetic screens for an
overwhelming number of different physiological processes.
Not only can mutations in critical genes be uncovered, they can
be maintained in populations through the use of balancer
chromosomes (chromosomes with multiple small inverted regions that prevent correct alignment of the chromatids and thus
crossing over). Balancer chromosomes are homozygous lethals, and carry easily scored phenotypic markers so that the
chromosome carrying the mutation can be identified.
Another advantage is that signaling pathways and interacting
factors of lethal mammalian genes may be elucidated in Drosophila by targeting their expression to a nonvital tissue, such
as the eye. The Drosophila researcher is fortunate enough to
employ several unique tools in the scientific toolbox (Supplemental Table S1). These include an array of aneuploid strains
(lines carrying chromosomes with deficiencies or duplicated
regions), classical mutations, and transgenic lines, including
Gal4/UAS and later systems generated along the same concept.
The Gal4/UAS system is bipartite: a line encoding the yeast
transcription activator protein Gal4 is crossed with a UAS line
carrying the Upstream Activation Sequence, which is an enhancer to which Gal4 specifically binds to activate gene transcription, and the progeny then expresses the gene downstream
of the UAS at a time and in the tissue(s) directed by the
promoter subcloned upstream of Gal4 (Fig. 2) (12). The majority of Gal4 lines are generated by constructs derived from
0363-6119/13 Copyright © 2013 the American Physiological Society
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Neckameyer WS, Argue KJ. Comparative approaches to the study of physiology:
Drosophila as a physiological tool. Am J Physiol Regul Integr Comp Physiol 304:
R177–R188, 2013. First published December 5, 2012; doi:10.1152/ajpregu.00084.2012.—
Numerous studies have detailed the extensive conservation of developmental
signaling pathways between the model system, Drosophila melanogaster, and
mammalian models, but researchers have also profited from the unique and highly
tractable genetic tools available in this system to address critical questions in
physiology. In this review, we have described contributions that Drosophila
researchers have made to mathematical dynamics of pattern formation, cardiac
pathologies, the way in which pain circuits are integrated to elicit responses from
sensation, as well as the ways in which gene expression can modulate diverse
behaviors and shed light on human cognitive disorders. The broad and diverse array
of contributions from Drosophila underscore its translational relevance to modeling
human disease.
Review
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bone development, and cardiogenesis, in addition to other
crucial developmental pathways (reviewed in Ref. 6).
In this review, we offer up only a limited number of major
contributions made by Drosophilists to understanding the dynamics of pattern formation, cardiac function, thermal and pain
sensation, and the basis of specific behaviors, including sleep
and learning. We have included a table listing recent reviews
for several research areas (Supplemental Table S2), but only
these few are presented in greater depth in this review. We are
limited not by the contributions, but by page constraints, with
the hope that the reader will come to appreciate how much
clinically relevant information about fundamental physiological processes has been derived from the small and unassuming
fruit fly.
Fig. 1. Life cycle and comparison with two vertebrate models. A: life cycle of
Drosophila melanogaster. B: comparative length of time per generation and
relative cost to maintain the same number of animals between Drosophila,
zebrafish, and mouse.
specific promoters or by fragments that randomly “trap” spatially and temporally specific enhancers upstream of Gal4
driven by a minimal promoter. Not only can a gene be expressed outside of its normal milieu, its expression may be
reduced via the use of RNAi transgenes, or monitored through
the use of reporter transgenes, and exogenous genes may be
expressed to establish they are functional orthologs of their
Drosophila counterpart (see Supplemental Table S1). While
only some of the vast array of tools are described in
Supplemental Table S1, more can be found on FlyBase
(www.flybase.org), a free website with links to multiple
stock centers (Bloomington, IN, USA; Kyoto, Japan; and
Vienna, Austria), other resources (Drosophila Genomics
Resource Center, Berkeley Drosophila Genome Project),
and, for all annotated genes (the vast majority), links to
sequence, predicted protein, expression profiles, interaction
pathways, available lines, and references.
The beauty of the unbiased genetic screens possible in this
system is that genes isolated based on seemingly unimportant
phenotypes were later demonstrated to be key players in the
etiology of human disease. For example, Notch, first identified
in Drosophila in 1915 as a wing mutation, has since been
shown to be the cause of human T-lymphoblastic neoplasms.
Notch is also critical for neurogenesis, neuronal differentiation,
Female Drosophila lay their eggs on or near a food source
for the hatching larvae to feast upon, and then fly away. While
humans, in general, nurture their offspring until mature, their
embryos also require factors to initiate the developmental
program. In flies, such factors are maternally deposited in the
nurse cells of the oocyte (hence their designation as maternal
effect genes); their role is to activate the first wave of zygotically expressed genes (gap genes). However, these genes are
known to be critical for organogenesis, in general, and the
detailed genetic characterization and expression profiles for
some of these genes have enabled researchers to use mathematical modeling to establish how dynamic pattern gradients
are formed, which is critical to our understanding of organogenesis and the physiological pathways relevant to individual
organ systems.
The first zygotic genes expressed in the developing embryo subdivide it into broad regions: terminal, “head,” and
central (reviewed in Ref. 101); mutations in these genes
create “gaps” by deletion of specific segments. The earliest
expression patterns of the gap genes are independently
determined by the maternal effect genes, but later expression
Fig. 2. The Gal4-UAS bipartite transgenic system. A line encoding the yeast
transcription activator protein Gal4 is crossed with a UAS line carrying the
Upstream Activation Sequence; Gal4 specifically binds to this sequence to
activate gene transcription. The temporal and spatial expression is directed by
promoter or enhancer sequences upstream of Gal4, and the progeny will
correspondingly express the gene downstream of the UAS sequences.
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Organogenesis, Gradients, and the Mathematical Dynamics
of Pattern Formation
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DROSOPHILA AS A TOOL TO UNDERSTAND PHYSIOLOGICAL PROCESSES
gradient maintained, since the integrity of the gradient is
obviously crucial for the next developmental stages? While
spatial distribution of bcd mRNA is clearly important, protein
movement is also necessary to establish the gradient (75), as is
degradation of Bcd protein via targeted ubiquitylation (77).
Although numerous models have been proposed to explain
how the gradient is established, none completely explain all the
experimental observations, a demonstration of the incredible
complexity of this critical biological question (45, 98). Just as
gap genes have been used as a tool to assess the mathematical
dynamics of pattern formation, bcd has served as a model for
mathematical modeling of a morphogenetic gradient (60).
While bcd is essential for anterior-posterior patterning of the
embryo, Dorsal (Dl) is key for dorsoventral patterning. As for
bcd, expression patterns of the multiple genes regulated by Dl
have been extensively characterized. Computational modeling
of the Dl gradient suggests its pattern is dynamic, with increasing amplitude but constant shape—very different from that
observed for the bcd gradient, which is expressed at the same
developmental time (65).
Since the mRNA and protein expression patterns for the
gap-gene network in Drosophila have been so extensively
detailed, mathematical models have also been constructed to
define the temporal embryological topology of these genes.
While some limitations obviously exist (i.e., assuming only
one enhancer per gene), such models can reasonably predict at
least some of the interactions between genes that are critical for
anterior-posterior patterning (9). These kinds of computational
approaches to predicting interacting genes within a network are
almost impossible to attempt in other species.
Drosophila as a Model for Human Cardiac Physiology
The heart, or dorsal vessel in Drosophila, functions to pump
hemolymph from posterior to anterior, and consists of cardiomyocytes surrounded by noncontractile pericardial cells. One
feature that makes this an ideal system for the study of both
cardiac development and physiology is that the dorsal vessel
can be visualized using only a light microscope during both
larval and adult stages, which makes it possible for researchers
to observe physiological parameters in the intact animal. This,
combined with the many genetic tools available in Drosophila,
has made it possible to identify and characterize many of the
key factors in cardiac development. One of the best known
examples of a cardiogenic factor identified by a candidate gene
approach in Drosophila is tinman (tin), which was named as a
result of the absence of a dorsal vessel in tin mutants (11). As
has been the case for many of the cardiogenic factors identified
in Drosophila, impairments in NKX2–5, the human homolog
for tin, results in congenital heart disease (107). This speaks to
the translational value of the Drosophila dorsal vessel, since so
many of the genes involved in cardiac development are conserved between vertebrates and invertebrates, and the resulting
defects in cardiac physiology as a consequence of misexpression of these genes are also similar (reviewed in Refs. 10 and
93) (Supplemental Table S3).
One of the most notable differences between Drosophila and
mammals is that the dorsal vessel is part of an open circulatory
system. This difference actually works to the advantage of the
Drosophila investigator, because an open circulatory system
allows for some transport of hemolymph even without a fully
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is dependent on the other gap genes, establishing multiple
gradients to define the individual domains (reviewed in Ref.
60). The gap genes encode transcription factors, and while they
are key players in early development, they are also necessary
for organ development. However, much remains to be elucidated to understand the molecular mechanisms guiding their
function. It has been proposed that the ancestral role of the gap
genes may be in neurogenesis and head patterning (60), but
they have also proven invaluable in increasing our understanding of the evolution of gene networks.
A mutation in a homeotic gene causes a given morphological
structure—like an antenna—to develop into a copy of a structure that belongs elsewhere on the body, like a leg. These are
the genes responsible not only for transforming segments into
body parts, but also for organogenesis, in general. The homeotic genes in Drosophila are divided into two clusters: the
Antennapedia cluster (ANTP-C), and the bithorax complex
(BX-C). The ANTP-C genes specify how the thorax and
abdomen of the fly are constructed (74). The proteins encoded
by the homeotic genes are transcription factors that activate
diverse downstream targets termed realizator genes, which,
when first described, were proposed to mediate cell adhesion,
polarity, and shape (40). The first description of the network of
realizator genes controlled by a homeotic gene (in this case,
Abdominal-B) identified targets such as GAP and GEF cytoskeleton regulators, cell adhesion molecules (e.g., E-cadherin)
and Crumbs, which is critical for determining apical-basal
polarity in the cell. While the individual organ structures will
not necessarily be conserved between Drosophila and humans,
similar networks of homeotic gene-controlled cascades likely
determine the changes in groups of cells that result in formation of specific organs.
Characterization of one of the critical maternal effect genes,
bicoid (bcd), has been fundamental to elucidating how expression gradients are established. Embryos derived from bcd
females lack anterior structures; normally, Bicoid protein is
expressed in a gradient with the highest concentration in the
anterior of the embryo. The protein is not detected in oocytes,
evidence that the maternally deposited mRNA is translationally
regulated (24, 36). Several elegant studies have demonstrated
that maternally derived bcd mRNA is anchored to the anterior
pole of the oocyte by virtue of specific sequences in its 3=
untranslated region via the actions of the products of several
other genes (8).
Using fluorescently tagged protein, Gregor et al. (44) have
shown that the gradient of Bicoid protein is established within
90 min. The gradient is remarkably precise from embryo to
embryo, since, in larger embryos, the gradient is appropriately
scaled up. These studies of bcd were the first to demonstrate
that positional information derived from a concentration gradient could establish polarity within an embryo and has made
a seminal contribution to our understanding of pattern development.
Bcd also acts as a downregulator of maternally expressed
caudal protein by binding to its mRNA via the homeodomain
in Bcd. caudal (cad) is required for posterior development of
the embryo and forms a posterior-to-anterior gradient; the
action of Bcd inhibits cad translation in the anterior half of the
embryo (102, 103).
How does Bcd stay concentrated in the anterior portion of
the embryo, instead of diffusing throughout; that is, how is the
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Fig. 3. Heart rate decreases with increasing age in a sexually dimorphic
manner. The heart rates of age-matched male and female wild-type (Canton S)
flies were compared over time. n ⫽ 40 individuals for each age (1, 3, 5, 10 or
15 days old). Individual heart rates were the average of 5 intervals of 15 s each
with 15 s between each interval. Statistically significant differences indicated
by asterisks over lines covering the bars. ***P ⬍ 0.001, Students t-test.
dimorphic. These changes with increasing age have been
correlated with alterations in the contraction pattern, suggesting that the likelihood of arrhythmias increase with increasing
age (91). Age-related cardiac decline has been linked to decreasing levels of the Drosophila ortholog of human KCNQ1,
which encodes a potassium channel ␣-subunit, and of dSur (an
ATP-sensitive potassium channel) (91). In humans, KCNQ1
alterations have been linked to an increased risk for arrhythmia
(see Ref. 61 for review). In flies, KCNQ has been shown to be
responsible for the slower repolarizing current, and its overexpression in older flies reduces the number of arrhythmias (91).
Similarly, it appears that in both flies and mammals, dSur
protects against pacing stress and hypoxic stress (2, 46). Lastly,
for flies, as well as for mammals, exercise training reduces
age-related cardiac decline (97, 113).
Insulin-IGF receptor (InR) signaling has a known role in
controlling life span (see Ref. 115 for review), making the
aging dorsal vessel an ideal model for studying organ-specific
aging in response to manipulation of insulin signaling. Mutant
lines with defects in the insulin-like receptor (InR) or its
substrate chico have an increased life span (19, 114). Direct
manipulation of insulin signaling in the dorsal vessel by overexpressing the phosphatase dPTEN (a negative regulator of insulin/IGF signaling) or the forkhead transcription factor dFOXO (a
negatively regulated target of insulin/IGF signaling), prevents the
decline in cardiac performance that is typically seen with increasing age, indicating that decreased insulin signaling not only
increases life span, but also specifically prevents age-related organ
decline (119).
Beyond its value as a model for cardiac aging, the dorsal
vessel in Drosophila can be used to evaluate general cardiac
dysfunction. Drosophila has homologs for all six cardiac
disease genes examined in a survey of 287 human disease
genes (34). In addition, many of the proteins needed to carry
out cardiac function in vertebrates, such as ion channels and
contractile proteins, are conserved (91). Mutations in several of
the ion channels and transporters have been found to alter heart
rate. Specifically, mutations in SERCA, the sarcoendoplasmic
reticulum Ca2⫹-ATPase, which has been linked to cardiac
malfunction in mammals, significantly reduces beat frequency
and alters electrical activity and rhythmicity (106). Mutations
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functioning dorsal vessel, making it possible to identify roles
for cardiogenic factors that would otherwise result in lethality.
Additionally, Drosophila has less genetic redundancy than
vertebrate systems, which can assist in the identification of
novel cardiogenic factors. Together, these features have facilitated the identification of novel genes involved in cardiac
development, elucidated complex interactions between multiple cardiogenic factors, and resulted in the creation of models
with translational value to mammalian cardiac physiology.
Following the initial development of the larval heart, some
additional modifications take place during metamorphosis to
create the adult dorsal vessel. One of the most important
differences between the larval and adult dorsal vessels is that
the adult dorsal vessel receives direct neuronal input, which
increases complexity and allows for adaptation to different
conditions (26). Importantly, many of the ion transporters
required for contractility of the heart in vertebrates are also
present and function in the dorsal vessel (76).
Several methods have been developed for high throughput
and translational cardiac analyses. One of the first studies to
look at the age-related decline of cardiac function in Drosophila used a light microscrope and video recording to measure
heart rate and end systolic and end diastolic diameter in
response to stress at different ages (95). By inducing heart
failure via external electrical pacing, it was found that as flies
age, maximal heart rate decreases, similar to what is observed
in humans (32, 95). Green fluorescent protein can be used in
conjunction with the Gal4/UAS system to allow for visualization and identification of specific cell types within the dorsal
vessel (e.g., 95). For continuous analysis, partially dissected
preparations in artificial hemolymph have been used to measure tension generated by the cardiac muscle using carbon
fibers (91). Analysis of video traces from these types of
preparations, as well as from intact preparations, can offer
several useful parameters, such as heart rate, systolic and
diastolic intervals, systolic and diastolic diameters, percent
fractional shortening, contraction wave velocity, and cardiac
arrhythmicity (31). An additional technique that involves enclosing a single fly in a small chamber to prevent large
movements and detecting the internal space of the heart tube
using optical coherence tomography has been used to establish
Drosophila as a useful model to study dilated cardiomyopathy
(121). For the study of electrical activity, recordings of intracellular action potentials can be performed even as the heart
contracts (71). One caveat for these types of studies is that care
must be taken to use a medium that mimics physiological
conditions specific for hemolymph (111). A video demonstrating the preparations and uses for several of the above mentioned techniques can be found at http://www.jove.com/index/
details.stp?id⫽1596 (20).
The Drosophila system offers several advantages that make
it attractive to modeling cardiac diseases beyond the ease with
which one can manipulate cardiac gene expression in vivo.
These above techniques, along with the advantage of a short
lifespan, have made Drosophila ideally suited to study cardiac
aging. Several similarities exist between the aging dorsal
vessel and the mammalian heart. Figure 3 shows that for both
male and female Drosophila, there is an overall trend toward
decreasing heart rate with increasing age, similar to what is
observed in mammals (32). Our analysis of heart rate in the
aging fly also shows that the changes with age are sexually
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DROSOPHILA AS A TOOL TO UNDERSTAND PHYSIOLOGICAL PROCESSES
Transient Receptor Potential Channels, Pain,
and Thermal Sensation
Every organism must sense and respond to deleterious
changes in their environment to survive; these include changes
in temperature, gustatory responses, vision, olfaction, and pain.
Not surprisingly, the neuronal mechanisms in place to sense
environmental disruptions are highly conserved across species.
While channels necessary for sensing these alterations have
been extensively characterized in the nematode, C. elegans,
and several have also been cloned from humans, what Drosophila has been able to offer is an understanding of how the
circuit is integrated, from perception to sensation to response.
It is critical for organismal survival to maintain an appropriate internal body temperature, as well as to seek out a
suitable external thermal environment; thus, all organisms tend
to actively avoid deleterious thermal conditions. There is a
temperature optimum for the normal regulation of cellular
physiology, such as membrane fluidity and biochemical reactions, which have evolved to function best within a limited
temperature range. Unlike mammals, which maintain a fairly
constant internal temperature, Drosophila (and other ectotherms) are dependent on the external environment to regulate
their internal temperature, and rely substantially on behavioral
mechanisms to maintain an appropriate body temperature
(110). It is not unreasonable to expect that the mechanisms for
sensation of different thermal conditions are ancient, since this
is a fundamental physiological process necessary for survival
and fitness.
While adult Drosophila can subsist between the temperatures of ⬃16°C and 30°C, they flourish at the mean of 23–
25°C. Since flies are small, they readily equilibrate to the
surrounding temperature. Therefore, if flies are to maintain an
appropriate body temperature for normal physiological processes, they must have evolved a rapid thermal sensing capability. And since they have two very different life stages that
must address the same problem, elucidating the mechanisms
for larval and adult thermal regulation should yield information
not only about thermal sensors in general, but about the
evolution of thermal regulation.
However, before one can understand the molecular mechanisms underlying thermosensation, it is necessary to identify
the temperature-sensing neurons and the organs in which they
reside, and this has proven to be comparatively facile using the
Drosophila model system. One of the first studies to identify
thermoresponsive cells and organs made use of transgenic
Drosophila expressing cameleon 2.1, a calcium-sensitive protein with two fluorophores whose fluorescence ratio changes
with a conformational change in the protein (e.g., when there
are changes in the intracellular calcium concentration). Cameleon 2.1 was expressed in all neurons in Drosophila larvae,
which were then exposed to differing cold and warm temperatures. These experiments identified neurons in the terminal
organ in the larval head that acted as cold sensors, and specific
body wall neurons that acted as heat sensors; this was confirmed using electrophysiological techniques (78). Additional
heat sensors were found to reside in central neurons (see
below).
The navigational decisions made by Drosophila larvae upon
encountering even small changes in temperature (0.005°C/s
gradient, 81) have been elegantly teased out. Drosophila larvae
crawling on a substrate frequently stop and “sweep” their heads
from side to side and then choose to orient along a specific
thermal gradient. This occurs via negative and positive thermosensors (similar to mammalian thermosensation) (reviewed
in Ref. 42), and downstream neural circuits integrate the input
from one set of sensory neurons to initiate turning or aligning
behavior (81). These experiments were some of the first to
elucidate the stochastic mechanisms underlying a goal-directed
behavior requiring executive decisions.
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in the Ca2⫹ channel encoded by cacophony results in an
abnormally fast heartbeat that does not change in response to
temperature (100). ORK1, an outward rectifying potassium
channel, was shown to regulate the pacemaker frequency in
Drosophila (71). This was the first evidence that a two-pore
domain potassium channel could regulate automatic cardiac
activity. Inactivation of ORK1 increases heart rate, and its
overexpression abolishes the heartbeat (71). Recently, a Drosophila model for atrial fibrillation was established using
tachypacing to induce cardiomyocyte remodeling, which
causes atrial fibrillation in humans (124), enabling researchers
to harness the power of Drosophila genetics to search for novel
targets for cardiomyocyte remodeling (124).
Hypoxia has been another physiological problem for which
Drosophila is ideally suited to aid in research. Hypoxia is a
major concern for human health because it is the leading cause
of necrotic cell death in myocardial infarction, as well as
having a fundamental role in embryogenesis, obstructive sleep
apnea, and sickle cell anemia. Adult Drosophila are tolerant to
low oxygen environments and can withstand anoxia for ⬃3 to
4 h without experiencing cell damage (48). Genes important in
hypoxia were discovered by exposing Drosophila to either
severe intermittent (present in humans during obstructive sleep
apnea, central hypoventilation syndrome, and intermittent vascular occlusion in sickle cell anemia) or constant (present in
humans during pulmonary diseases such as asthma and congenital heart disease with right to left shunt) hypoxic conditions, and performing microarray analysis to identify up-regulated or downregulated transcripts (3). Overexpression of
Hsp70 and Hsp23 during constant hypoxia, and Mdr49 and
l(2)08717 during intermittent hypoxia, increased survival (3).
Other correlates have been discovered: for example, trehalose,
a glucose dimer, plays a role in protecting both fly and
mammalian cells from anoxic stress. Overexpression of trehalose-6-phosphate synthase 1 (tps1), which synthesizes trehalose, increases tolerance to anoxia in flies, and disruption of the
tps1 gene results in lethality (15). Increased resistance to low
oxygen stress has been observed in mammalian human embryonic kidney cells transfected with Drosophila tps1 (16). Hypoxia-inducible factor, the metabolic AMP-activated protein
kinase, and nitric oxide signaling have all been shown to be
involved in the response to hypoxia in the mammalian heart
(62, 67, 83); not surprisingly, all are present and functional in
flies (72, 94, 120). Feala and colleagues are compiling data
from the annotated Drosophila genome (1), the KEGG database of enzymes and pathways (92), and Drosophila biochemistry data from the literature to build a database of all metabolic
pathways in Drosophila myocytes (28) to create a genomewide model that includes all genes, enzymes, metabolites, and
regulatory proteins, as well as protein-protein interaction pathways, that are involved in the hypoxia defenses (29).
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dTRPA3, also known as water witch, functions as a hygroreceptor to detect humidity (79). A second TRP channel,
nanchung (a TRPV channel like inactive, below), detects dry
air; interestingly, the compartmentalization of sensation for
detecting moist vs. dry is similar to the compartmentalization
of receptors that detect cold vs. heat.
Several “cold” larval receptors in Drosophila have also been
described; these include the TRPC channels TRP and TRPL,
which detect relatively small differences in temperature (105).
Larvae carrying a genetic disruption in each of the 13 TRP
channels were tested in a simple choice assay (between 14°C
and 17.5°C); this screen identified the two TRPV channels
(70). The TRP channel Iav was shown to function in thermal
discrimination of cooler temperatures in the chordotonal organs by genetically targeted disruption, resulting in increased
turning angles of the larva along the substrate (70). Adult cold
sensors have been identified in a simple adult behavioral screen
of insertional mutations (fly strains in which a small transposable element was inserted within or adjacent to a gene, disrupting expression of that gene) (39). This screen uncovered
brivido (brv)—Italian for shiver—a member of the TRP polycistin subgroup of the TRP ion channel family. A second TRPP
mutation, brv2, was identified by homology; flies lacking brv2
function also display impaired avoidance of cold (as low as
11°C) temperatures. A third TRPP-homologous gene, brv3,
was similarly shown to act as a cold thermosensor. The three brv
genes are coexpressed in the same three neurons within the arista
(part of the fly antenna) and serve as cold sensors; the other three
aristal neurons are heat sensors. Tracking the projections from
the aristal neurons to the brain (using a targeted green fluorescent protein, and a few genetic tricks to specify projections
from only the cold or the heat thermosensors) demonstrated
that they converged onto adjacent but distinct glomeruli (39).
These elegant experiments demonstrated that distinct cell populations coordinate behavioral responses to specific temperatures.
The TRP family members discussed above were all initially
identified years before their human homologues (Supplemental
Table S4). In some cases, mutations were characterized long
before their functions in pain or thermal sensation were recognized (i.e., trp, trp-like [trpl] and inactive [iav]). Additional
TRP family members were isolated by homology, or by simple,
high-throughput behavioral screens, or by a combination of
screening only those lines carrying mutations in genes known
to be homologous to the TRP channel family. In fact, the
human TRP homolog was identified via homology with the
Drosophila trp gene (118).
While the TRP channels are key in thermal sensation, they
are not, however, the whole story. Another forward genetic
screen, using the same 27,000 insertion lines mentioned above,
identified elements of the histamine signaling pathway, including histidine decarboxylase and two histamine receptors, as
demonstrating a low tolerance for high temperatures, and
increased tolerance for cold (57). Independent confirmation
was achieved using known mutant alleles corresponding to
these genes. In addition, treatment with histamine receptor
antagonists was shown to affect temperature preference. Histamine is found in a limited number (⬃20) of neurons, but
arborizations from these cells are heavily distributed throughout the adult brain; the receptors are also widely expressed in
neural tissue (57). These studies demonstrated a novel role for
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“Heat” signals are transduced, at least in part, by members of
the TRP family, which share homology with the mammalian
TRP family genes. TRP channels are ancient, with extensive
functional conservation amongst the subfamily members (86).
The first TRP channel identified in Drosophila, painless, is
found in neurons within the larval body wall, and is activated
by high heat (⬎42°C, well outside the fly’s optimal temperature, and thus considered noxious) (116); it is also activated in
response to mechanical insult. Consistent with this phenotype,
painless is expressed in the multidentritic neurons in the
periphery (116). painless is the Drosophila ortholog of the
mammalian wasabi receptor, TRPA1, and was isolated by
screening an insertional mutation library for animals that did
not produce the stereotypical kink-and-coil reaction in response to the touch of a hot (⬃40°C) probe. It shares homology
with a member of the TRPN subfamily, no mechanoreceptor
potential-C (NOMP-C), which is also required for mechanosensation. The isolation of painless is yet another testimony
to the utility of the Drosophila genetic screen: 1,500 lines were
screened in short order and yielded 49 candidates with decreased sensitivity to noxious heat, of which painless was the
first one characterized.
There are three other members of the TRPA subfamily:
dTRPA1, dTRPA2, and dTRPA3. dTRPA1 is also activated by
higher temperatures and is found within specific cell clusters in
the larval central nervous system, as well as in cells found
within the corpus cardiacum of the ring gland, the fly’s endocrine organ, and in cells dispersed throughout the gastrointestinal tract. However, it is only the anterior cell neurons in the
brain that are required for thermal sensation (49). This channel
modulates selection of an environmental temperature of
⬃18°C when the animal is exposed to only slightly warmer
(⬃24°C) conditions (69). Recently, the G protein-coupled
receptor necessary for thermosensory signaling by dTRPA1
was shown to be a rhodopsin-ninaE, acting in a pathway
independent of any activation by light (108). These studies
determined that Gq, phospholipase C, and TRPA1 are downstream elements in the pathway initiating with RH1, the opsin
encoded by ninaE; more importantly, and again demonstrating
conservation across species, the human ortholog (melanopsin) can
rescue the thermal sensory defects in ninaE mutant larvae (108).
How do TRPA1 channels discriminate between thermal and
sensory cues? Such specificity may be established by selective
expression of different isoforms: TRPA1(A) is insensitive to
warmth and is expressed in gustatory neurons; the chemically
sensitive TRPA1(B) isoform is instead expressed in these cells
(64). This is specified by a unique region within the B isoform
that significantly reduces its thermosensitivity (64).
dTRPA2 was isolated from an insertional mutagenesis
screen of more than 27,000 lines. Known as pyrexia (a medical
term meaning fever), it is expressed in both multidendritic and
nondendritic cells lining the larval epidermis; in adults, it is
expressed in sensory neurons underneath the eye bristles, and
in neurons innervating the back of the thorax, maxillary palps,
proboscis, and antennae. In all cases, it is expressed throughout
the neurite, suggesting it functions in body-wide responses.
Channels encoded by pyrexia are involved in the sensation of
noxious heat, and animals carrying a mutation in this gene
become paralyzed after a short exposure to noxious (40°C)
heat (73).
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DROSOPHILA AS A TOOL TO UNDERSTAND PHYSIOLOGICAL PROCESSES
Using Drosophila to Elucidate Biobehavioral Mechanisms
Aggression. Aggression is a complex behavior seen in almost all social species, including Drosophila. It has many
purposes, but often in the animal kingdom it plays a role in
territory defense, survival or reproduction, and is also observed
in psychiatric disorders in humans. The first account of aggressive behavior in Drosophila came from Jacobs, who described
males fighting for food sources (59). Drosophila has since been
used to identify several genes involved in aggressive behavior,
which may have implications for aggression in humans. Many
of the studies that have been done to date have focused on
selectively breeding for males that display increased aggressiveness, are more territorial, and have greater success when
fighting other males, and identifying upregulated and downregulated genes relative to wild-type in these selected populations (22, 27). Several of the genes found to be associated with
altered aggressiveness in males have been previously identified
to have roles in a wide range of biological processes, including,
sex determination, neurotransmission, learning and memory,
pheromone detection, and nervous system development (see
Ref. 104 for review). Importantly, many of these genes also
have human homologs (29).
In addition, several studies have assessed the role of biogenic amines in aggression. Baier et al. (5) showed that
octopamine, dopamine, and a region of the adult fly brain
known as the mushroom bodies (analogous to the mammalian
hippocampus) all show reduced levels of aggression (5).
Sleep. Although the exact purpose of sleep is unknown, it is
conserved in all animal species that have been examined thus
far (18). Drosophila, like all other animals studied, demon-
strate a cycle of ⬃24 h even when separated from external
cues, such as temperature and light, and there are distinct
periods of sleep and rest, demonstrated by differing levels in
activity. One of the best known contributions from Drosophila
to the study of sleep was the discovery of the clock genes
(reviewed in Ref. 96). The first clock gene was discovered by
identifying flies with abnormal circadian patterns of eclosion,
and it was named period (68); years later, it was found that
period has homologs in many other species, including some
mammals (96).
Sleep in a fly is defined as any period of immobility lasting
for more than 5 min, which shares most of the features of sleep
in humans (53, 58). In flies, it is possible to measure sleep
depth by looking at parameters such as sleep continuity (58).
Electrophysiological recordings can also be performed, but
these are generally invasive (89). Many studies have focused
on identifying those genes involved in sleep in the hope that,
by understanding the biological processes modulated by these
genes, the reason for sleep will be revealed.
Another well-known example is the forward genetic screen
that identified Shaker, which selected for short-cycle sleeping
mutants and identified minisleep, a Shaker allele. Shaker encodes the ␣-subunit of a voltage-dependent potassium channel,
which is involved in membrane repolarization and transmitter
release (17). Knocking out Kcna2, the mouse Shaker ortholog,
also results in reduced sleep (23).
Reverse genetic screens have also been used to further
investigate the role of genes and pathways already known to be
involved in sleep. For example, previous studies had shown
that the cAMP/PKA/CREB pathway is important for learning
and memory, and that CREB expression displays a circadian
pattern (7). Increasing cAMP or CREB activity in Drosophila
decreases sleep time and rebound after sleep deprivation (54);
later studies in mice showed similar results (43).
Drosophila as a model to study learning and memory.
Investigation of the neurological processes underlying learning
and memory may provide progress toward elucidating the
etiologies of human cognitive disorders. In addition to the
genetic screens that have repeatedly proven to be beneficial,
there are also numerous behavioral assays developed for Drosophila that have facilitated a greater understanding of the
molecular mechanisms underlying learning and memory. The
first technique to be used was based on a fly’s ability to learn
to avoid an odor associated with an electric shock (99). Since
its initial development, the odorant learning method has been
modified and generates a remembered association that can last
up to a week (117). Odorant learning methods have been used
to identify several genes involved in learning and memory that
are generally expressed in the mushroom bodies and central
complex, two structural regions within the Drosophila brain
that mediate higher-order behaviors (51, 52). Another popular
learning paradigm is conditioned courtship suppression, which
is based on the fact that mated females will block male
copulation attempts. Rejected males will eventually learn to
suppress their courtship behavior toward other females (109).
This paradigm is less widely used for large-scale screens than
the odor association method, since courtship studies are more
tedious and limited to males. A parallel technique exists that is
based on the courting of newly eclosed males by older males.
Since the courtship attempts in this case will not end in
copulation, the older male eventually learns that his attempts
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histamine beyond its functions in phototransduction, mechanosensation, and sleep, and they suggest a possible involvement in seasonal acclimation that may occur with changes in
daylight and temperature.
Similar to human neuropeptide Y, the Drosophila homolog,
neuropeptide F (NPF), has been shown to play a role in
nociception by modulating TRP channel-mediated responses to
noxious heat stimuli (123). A G protein-coupled NPF receptor
is coexpressed with painless in peripheral thoracic neurons;
additional experiments have shown that expression of the rat
capsaicin receptor TRPV1 in neurons expressing painless resulted in larval aversion, which was rescued by coexpression of
NPRF1 (123). These studies have very elegantly demonstrated
a conservation of mechanism across species.
Thus, through the use of extensive genetic screening, multiple genes in different pathways have been identified as being
critical for thermal sensation and pain. In addition, the subpopulations of cells, in different tissues and organs, necessary
for perceiving these signals, as well as some of the molecules
required for their transduction, have been characterized in an
effort to integrate these pathways. The beauty of the model is
that a simple assay—alignment along a small thermal gradient,
or reduced aversion to a noxious stimulus—which takes only a
few minutes to complete, has enabled researchers to begin to
piece together the step-wise process of an evolutionarily conserved goal-directed behavior. And of course, acknowledging
the conservation across species, the first mammalian TRP
family members were isolated by using the Drosophila gene as
a stepping-stone.
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starvation resistance, it has been shown that it is most commonly accomplished in Drosophila by increasing energy stores
(reviewed in Ref. 56). In addition, animals selected for starvation resistance had increased resistance to other stressors but no
increase in longevity (50).
Another commonly encountered stress is oxidative stress,
which can trigger numerous diseases, such as cancer, stroke,
Parkinson’s disease, cardiovascular disease, and many other
diseases of aging (reviewed in Ref. 33). In Drosophila, oxidative stress is easily accomplished by feeding the animals
paraquat or by genetic manipulations to the electron transport
chain, because mitochondria are the primary source of reactive
oxygen species. One study used paraquat to show that increasing oxidative stress acts to increase tau-induced neurodegeneration in Drosophila (21). Sun and colleagues found that
Fig. 4. Recruitment of dopamine neurons into the stress response circuitry is
dependent on sex and level of sexual maturity. A: sexually mature male and
females were assayed for time spent freezing following a 24-h exposure to
paraquat (oxidative) stress. Three-way ANOVA (genotype ⫻ sex ⫻ stress)
P ⬍ 0.001, two-way ANOVA (sex ⫻ stress) P ⬍ 0.05 for elavC155/w1118 and
P ⬍ 0.01 for 854/THK, ANOVA (stress) **P ⬍ 0.01, *P ⬍ 0.05. B: sexually
immature and mature females were assayed for frequency of freezing bouts
following a 24-h exposure to paraquat stress. Three-way ANOVA (genotype ⫻
age ⫻ stress) P ⬍ 0.001, two-way ANOVA, P ⬍ 0.05 for both, ANOVA
(stress) ***P ⬍ 0.001. elavC155/w1118, control; 854/THK, targeted knockdown
in dopamine synthesis in a subset of dopamine neurons within the mushroom
bodies. n ⫽ 45.
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are futile and reduces his courtship attempts (38). This technique was used to establish a role for dopamine in habituation
learning (87). An additional technique that is simple, useful for
both sexes, and sufficiently high throughput for use in a
large-scale genetic screen, is the use of a heat box. A heat box
is a small chamber into which a single fly is placed, and
permitted to roam freely, but when it crosses the midline of the
box, the temperature will be increased in the chamber, training
the fly to prefer the cooler side of the chamber (122).
Dunce (dnc) was the first of the learning mutants to be
discovered using the odorant learning paradigm. dnc mutants
displayed normal phototaxis, an ability to sense odors and
normal deterrence of electric shock (25), yet they were unable
to learn to associate a particular odor with an electric shock
(25). dnc was later found to encode cyclic AMP (cAMP)
phosphodiesterase II (13). An additional learning mutant, rutabaga (rut), was identified using the same behavioral paradigm as for dnc and encodes a Ca2⫹/calmodulin-sensitive
adenylyl cyclase (80). A third learning mutant, amnesiac,
encodes the pituitary adenylyl cyclase-activating peptide (30,
99a). The results from dnc and rut provide strong evidence that
regulation of Gs signaling and cAMP levels are essential for
normal learning and memory in Drosophila.
In addition, Drosophila models exist for many of the human
disease-associated cognitive deficits. Neurofibromatosis is a
dominant genetic disorder in humans characterized by benign
neuronal tumors and defects in movement and learning due to
mutations in Nf1 (see Ref. 63 for review). A Drosophila strain
lacking Nf1 was generated to help identify molecular pathways
involved in this disease (47). The authors found that Nf1 may
be involved in cAMP signaling by regulating activation of rut
(47). Drosophila has also been used to model tauopathies.
Overexpression of TAU in Drosophila results in deficits in
learning and memory (85). The most notable symptom of
trisomy 21 (Down’s syndrome) is mental retardation, believed
to be due to overexpression of DSCR1, a gene that encodes
calcipresin, an inhibitor of the serine/threonine protein phosphatase calcineurin (37). Overexpression of the Drosophila
ortholog, nebula, results in severe immediate memory and
consolidated memory defects with decreases in protein kinase
A activity and phosphorylated CREB, additional evidence for
the involvement of CAMP signaling in learning and memory
(14). Fragile X syndrome, characterized by mental retardation, is caused by disruption of the FMR1 gene (see Ref. 55
for review). Disruption of FMR1 in Drosophila results in
abnormal mushroom body development and defects in memory in the courtship conditioning paradigm (84). Glutamate
receptor antagonists and lithium can restore learning and
memory deficits in this model, which may be helpful for
human patients (84).
Stress. On a daily basis, animals of all species must deal with
changing physiological and environmental conditions. Starvation is a commonly encountered stress for a variety of species,
and prenatal malnutrition in humans has been linked to diseases, such as schizophrenia and other forms of psychosis
(reviewed in Ref. 82). Starvation stress is modeled in flies by
providing access to water but no food. One study showed that
starvation resistance is heritable by placing flies on a decomposing lemon, which has insufficient nutritional value, and
selecting for increased resistance to starvation (50). Although
there are a few ways in which animals may increase their
Review
DROSOPHILA AS A TOOL TO UNDERSTAND PHYSIOLOGICAL PROCESSES
Summary
The contributions of the Drosophila model to our understanding of numerous diverse physiological pathways cannot
be overemphasized. These studies also serve as a shining
example of the pivotal importance of unfettered basic research,
since so much of what we now realize to be of critical
significance in the etiology of human disease can trace its
lineage to genetic mutations identified in Drosophila.
Glossary
Central complex: an associated group of structures within
the insect brain critical for locomotion, coordination between
the two brain hemispheres, and some aspects of cognitive
behavior.
Chordotonal organs: the stretch receptor organs in arthropods that help detect vibrations caused by sound, or the
position of body appendages.
Eclosion: When the adult fly emerges from the pupal case
after metamorphosis.
Forward screen: a (generally unbiased) genetic screen to
identify genes based on a known phenotype. A reverse screen
begins with the mutation and identifies the associated phenotype(s).
Homeotic gene: genes critical in development, expressed in
specific patterns, and which, when mutated, result in the
transformation of one body region or appendage for another.
Mushroom bodies: structures within the insect brain composed of Kenyon cells and dense neuropil, considered the
analogue of the mammalian hippocampus.
Pupariation: formation of the pupal case by the larva during
metamorphosis.
GRANTS
This work was supported in part by R01MH083771 from the National
Institutes of Health to W. S. Neckameyer.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: W.S.N. and K.J.A. prepared figures; W.S.N. and
K.J.A. drafted manuscript; W.S.N. edited and revised manuscript; W.S.N.
approved final version of manuscript.
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