Download `Am not I a fly like thee?` From genes in fruit flies to behavior in humans

Human Molecular Genetics, 2004, Vol. 13, Review Issue 2
doi:10.1093/hmg/ddh248
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‘Am not I a fly like thee?’ From genes in fruit
flies to behavior in humans
Ralph J. Greenspan* and Herman A. Dierick
The Neurosciences Institute, 10640 John Jay Hopkins Drive, San Diego, CA 92118, USA
Received June 14, 2004; Revised and Accepted July 26, 2004
The fruit fly Drosophila melanogaster has provided insight into the role of genes in behavior, some of which
have relevant implications for humans. Mutants induced or engineered in the laboratory have contributed to
our understanding of biological rhythms, learning, memory, neurodegenerative disease and drug response.
Studies of naturally occurring genetic variation in behavior have advanced our understanding of what kinds
of variants arise spontaneously and contribute to behavior.
The Fly
Little Fly,
Thy summer’s play
My thoughtless hand
Has brushed away.
Am not I
A fly like thee?
Or art not thou
A man like me?
For I dance,
And drink, and sing,
Till some blind hand
Shall brush my wing.
If thought is life
And strength and breath,
And the want
Of thought is death;
Then am I
A happy fly.
If I live,
Or if I die.
from ‘Songs of Experience’
by William Blake
Charting the relationship between genes and behavior can be
a perilous activity. The challenges are many: the difficulty
of defining genetic and non-genetic factors, the difficulty of
understanding the interactions among these many factors,
the many steps that intervene between the expression of
a gene and the manifestation of a behavior, and the inescapable fact that each individual is the unique product of a series of
historical accidents. Historical uniqueness also applies to nonhuman biological individuals. It is a hallmark of the biological
world due to the fact that no particular combination of genes
and experiences is ever replicated exactly in nature, although
this is not equally apparent for all behaviors. Moreover, all of
these difficulties are compounded many times over by the
limitations on what kinds of experimental approaches can be
brought to bear on human behavioral traits.
Studies of genes and behavior in model organisms offer one
way out of this conundrum. To begin with, it is far easier to
control environmental conditions and genetic heterogeneity
in the laboratory, and such studies are generally undertaken
keeping environmental conditions as uniform as possible and
genetic makeup as well defined as possible. But this would
be no help if the phenomena and principles are fundamentally
different from those of humans. Fortunately, they appear to be
similar by an ever increasing number of criteria. One of the
most striking outcomes of modern biology and the sequencing
of genomes is the similarity of genes between distantly related
creatures. The fruit fly Drosophila melanogaster and humans
share a great many of the same genes in common. Similarly,
they share considerable conservation of metabolic and signaling pathways at the cellular level. Most strikingly, and most
relevant for this discussion, there is growing evidence for conservation at the level of behavior and its molecular mechanisms. This has been shown most convincingly, thus far, for
several behaviors in Drosophila: circadian rhythms (1), learning and memory (2) and most recently sleep (3). Thus, there is
good reason to believe that the characteristics, interactions and
*To whom correspondence should be addressed. Tel: þ1 8586262075; Fax: þ1 8586262099; Email: [email protected]
Human Molecular Genetics, Vol. 13, Review Issue 2 # Oxford University Press 2004; all rights reserved
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Human Molecular Genetics, 2004, Vol. 13, Review Issue 2
contributions of genes in the behavior of model organisms will
provide important guidelines for how to think about the role of
genes in human behavior.
More recently, there has also been a movement towards
creating Drosophila models for human diseases affecting the
nervous system, such as Huntington’s Disease, Parkinson’s
Disease and Alzheimer’s Disease. These have been created
not only for the purpose of trying to understand underlying
genetic and molecular mechanisms of the disease, but also
as a platform for drug screening. Similarly, the fly’s responses
to ethanol, cocaine and nicotine are also being studied as possible models for addiction.
As a consequence, the fruit fly has taken on some surprising
new roles as a potential model for human biology and genetics. All try to capitalize on the versatile genetic manipulations possible in the fly, and then look for transferability to
humans either at the level of fundamental mechanism, or at
the level of preliminary therapeutic screen.
FUNDAMENTALLY CONSERVED MECHANISMS
Circadian rhythms
The clearest contribution fruit flies have made is in unraveling
the cellular mechanism of the circadian clock. These discoveries began with the isolation of long-day, short-day and
arrhythmic mutants in Drosophila, all of which proved to be
alleles of the same gene, dubbed period (4). Through the isolation of additional mutants in flies, fungi and mice and the
cloning of these genes, the cellular mechanism of the circadian
clock was worked out and shown to be nearly universal in the
biological world (1,5,6). Many of the actual genes involved,
such as period, are conserved between flies and mammals
(Fig. 1).
From the discovery of the period gene, the quest progressed
with the isolation and cloning of additional mutants: timeless,
doubletime, Clock, cycle and cryptochrome (1). What has
emerged is a picture of the circadian clock as a transcriptional
regulatory loop in which each cell counts out its own 24 h
period by means of an oscillating cycle of transcription and
translation. Several other genes complete the basic cycling
mechanism—per and tim repress their own transcription which
requires activation by the transcription factors Clock and
cycle, with the doubletime kinase regulating the degradation
of period and timeless proteins (1). This serves as the central
time-keeping mechanism for all circadian rhythmic activities.
Aside from the very high degree of molecular conservation
of this mechanism in mammals, including humans, there have
also been two sleep syndromes linked to variants in human
homologs of the period gene. A haplotype of the human
period3 (hPer3 ) gene, one of the multiple human homologs
of Drosophila period, has been associated with delayed
sleep phase syndrome (7). A separate study of Finnish patients
with the same sleep syndrome revealed a length polymorphism in the hPer3 gene, such that the long allele favored
morning preference and the short allele favored evening
preference (8).
Another hereditary sleep disorder, familial advanced sleep
phase syndrome is an autosomal dominant circadian rhythm
variant in which affected individuals are ‘morning larks’
with a 4 h advance of the sleep, temperature and melatonin
rhythms. Affected individuals have a serine to glycine
mutation within the casein kinase Iepsilon (CKIepsilon)binding region of hPER2, which causes hypophosphorylation
by CKIepsilon in vitro. Thus, a variant in human sleep behavior may be attributable to a missense mutation in a clock
component, hPER2, which alters the circadian period (9).
Further contributions to the understanding of human sleep
are likely to emerge from studies in the new realm of fruit
fly sleep (3).
Learning and memory
Learning and memory have many features common to flies,
rodents and humans. Behaviorally, all are capable of associative conditioning, and the memory that is induced shows
multiple phases that can be distinguished by pharmacology
and by training regimen (2). In the fly, learning takes the form
of distinguishing between two odors and avoiding the one that
has previously been paired with electric shock. Long-term
memory is produced by repeated training trials separated by
suitable time intervals. Care has been taken in all searches for
learning mutants to ascertain that these flies are normal in all
other relevant faculties that are required for the learning task:
olfaction, locomotor activity and shock reactivity.
The original Drosophila learning mutant, dunce (10),
encodes one of two forms of cAMP-phosphodiesterase.
Mutations in other components of the cAMP signaling system,
rutabaga in the synthetic enzyme (adenylate cyclase), and
Dco and RI in the key enzyme regulated by cyclic-AMP
(cAMP-dependent protein kinase), as well as the Creb
(cAMP-response-element-binding) protein that regulates genes
induced by cAMP, were subsequently shown also to affect
learning and memory in the fly (Fig. 2) (2). Moreover,
various of these components had selective effects on different
phases of memory. The cyclic-AMP signaling system had
already been implicated in neuronal plasticity as central to
the mechanism that modifies efficacy at synapses in the circuitry underlying learning. The isolation of mutants affecting
the system from unbiased, open-ended mutant screens and
the demonstration that they affected classical conditioning
in the intact animal gave universality to the finding. These
findings not only placed the differential genetic effects on
memory phases on firmer ground, they were also subsequently
extrapolated to mice and rats (2). Subsequent mutant hunts for
selective effects on long-term memory have also revealed the
role of genes involved in mRNA localization and translational
control (11).
The relevance to humans has been borne out by the fact that
there is a hererditary disorder, Rubinstein – Taybi syndrome
(RTS), characterized by mental retardation and physical
abnormalities including broad thumbs, big and broad toes,
short stature, and craniofacial anomalies, that maps to chromosome 16p13.3, a genomic region containing CREB-binding
protein (12). More significantly, many RTS patients are heterozygous for CBP mutations that yield truncations of the
CBP C terminus. On the basis of the fly work, and its subsequent confirmation in mice, memory enhancement drugs
are currently being tested in clinical trials that target cAMP
phosphodiesterase (13).
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Figure 1. Conservation of molecular mechanisms of the circadian clock between flies and mammals. PER, period gene: TIM, timeless gene and CKIe, casein
kinase Ie gene. (Adapted with permission from 43. Copyright 2000 AAAS.)
Validity of fly models for circadian rhythms
and learning
The fundamental biological principles that have been obtained
from genetic studies of circadian rhythms and learning and
memory in the fruit fly have proved to be widespread, if not
universal, in the animal world. Most important for our discussion is that they have opened the way for understanding the
human biology underlying these behavioral phenotypes. This
is an incontrovertible argument for their relevance as models
for humans.
SYNTHETIC FLY MODELS OF HUMAN
PHENOTYPES
Neurodegenerative disease
The completion of the Drosophila genome sequence has made
it possible to identify genes in the fly that are homologous to
human disease genes (14 –17). Approximately two-thirds of
the known human disease genes have a counterpart in Drosophila (666 of 911 genes) (16,17) and 74 of these genes,
roughly 10%, are involved in neurological diseases. It is in
this area that the last few years have seen a veritable explosion
of research using the fly as a model for human disease. Most of
these models, however, have focused on three neurodegenerative diseases, Huntington’s, Parkinson’s and Alzheimer’s
disease involving mis-expression of the relevant mutant
human genes for huntingtin, a-synuclein and Ab1 – 42, respectively. The strategies to study these and other diseases in flies
cover the spectrum from basic biology to pragmatic pharmacotherapy. Consequently, some studies focus on the endogenous biological function of the homologous fly gene with little
reference to human disease, whereas others try to cure a simulated human fly disorder with the hopeful expectation that it
will translate to a successful treatment in humans.
A fly model expressing the mutant human huntingtin, the
human SCA3/MJD mutant gene or simple polyglutamine
repeats in the compound eye causes neurodegeneration of
photoreceptor neurons (18 –20). Screens for mutations in
other genes that modify this degenerative eye phenotype
have identified HSP70 and several other chaperones as protective components (18 –20). This seems significant, given
the presence of HSP70 in the protein aggregates characteristic
of the pathology of huntingtin –induced neurodegeneration,
suggesting that protein folding affects aggregate formation.
This protective effect seems to be conserved in other model
systems as well (18 – 20). The TOR inhibitor rapamycin has
also been shown to protect against polyglutamine-induced
degeneration in the fly eye (21). In one instance, that of
the SCA3/MJD mutant gene, the antiapoptotic gene bcl-2
was also shown to have a protective effect. Flies expressing
this SCA3/MJD mutant gene have been tested behaviorally
and shown to exhibit locomotor defects and anosmia (22).
A fly model of Parkinson’s disease has been created through
expression of wild-type or mutant forms of the human a-synuclein gene in the fly brain. Although flies have no endogenous
a-synuclein gene, its expression can recapitulate many aspects
of the disease in flies, including degeneration of dopaminergic
cells (18 –20). The age-dependent loss of locomotor control
seen in these engineered mutants can be ameliorated by some
of the same drugs used to treat human Parkinson’s patients:
L-DOPA and various dopamine agonists (including bromocryptine and SK&F 38393) ameliorated the deficits (23).
A Drosophila version of another Parkinson’s disease gene,
parkin, exists and a mutation at the fly locus causes mitochondrial swelling, muscle and locomotor defects, but does not lead
to a loss of dopmanergic neurons (19,20). Over-expression of
fly parkin partially suppresses the a-synuclein-induced
degeneration phenotype (19,20,24). As in the Huntington’s
fly, over-expression of HSP70 can also protect dopaminergic
neurons against a-synuclein toxicity (18 –20).
Flies have endogenous versions of APP (called APP-like
or Appl ) and of Presenilin-1, both implicated in familial
Alzheimer’s disease in humans. Fly Presenilin has been
shown to play a role developmentally through its effect on
cleavage of the NOTCH protein and work in the fly has
added greatly to the understanding of APP processing
(19,20). Fly Appl causes axonal vesicular accumulations
when over-expressed or when mutated, suggesting that the
normal protein plays a role in axonal transport (20,25).
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Potential value of fly models for neurodegeneration
Because the models for neurodegeneration are mainly synthetic, consisting of mutant human genes expressed in the
fly nervous system, their study departs from the sort of basic
biology described earlier. (Mutants such as spongecake, if
proven to be bona fide, endogenous biological models for
human neurodegeneration, would represent an exception.)
The behavioral defects seen in these models only vaguely
resemble the human symptoms. To the extent that the
perturbations of the fly’s biology and the synthetic strains
produced can be understood mechanistically, however, these
models may hold promise for identifying interventional
strategies to block or retard degeneration. The implication of
chaperone proteins as neuroprotectors against polyglutamineinduced degeneration, and of certain kinases and phosphatases
as neuroprotectors against tauopathy-induced degeneration
provides the best example thus far of the sort of potentially
useful information that such studies may yield.
Addiction and drugs of abuse
Figure 2. The cAMP signaling pathway in memory formation. AC, adenylate
cyclase (rutabaga gene); PDE, cAMP phosphodiesterase (dunce gene); PKA,
cAMP-dependent protein kinase (DCO and RI genes) and CREB, cAMPresponse-element-binding protein (dCreb gene). (Adapted from 44 with the
kind permission of Nature Publishing Group, http://www.nature.com/.)
Behavioral effects have been shown in the fly Appl knockout mutant and in flies expressing human Ab-peptides. The
Appl mutant causes a general non-reactivity to stimuli that
normally elicit phototaxis or chemotaxis, a phenotype that
can be partially rescued by expression of a normal human
APP gene (26). The strains expressing the Ab1 – 42 peptide
exhibit Alzheimer’s-like degeneration and deficits in learning
when the peptide is expressed in a part of the brain (the mushroom bodies) known to be required for associative conditioning (27).
Tauopathy can also be produced transgenically in the fruit
fly, most pronounced when a mutant human gene is used (28).
A screen for genetic modifiers of its neurodegeneration
yielded a set of kinases and phosphatases as prominent among
the enhancing and suppressing genes, contrasting with the
findings for polyglutamine-induced degeneration (see earlier).
Over-expression study of normal tau protein in the mushroom
bodies of the fly brain (29) produces learning deficits in the
absence of degeneration, suggesting that behavioral deficits
are a more sensitive indicator of malfunction than pathology.
In addition to the biochemical and apparent behavioral similarities of these fly models to human biology, there are also
similarities in histopathology between some fly neurodegeneration mutants and human disease. One such example is
shown in Figure 3, comparing ultrastructural phenotype
between the fly mutant spongecake and human Creutzfeldt –
Jakob disease.
The neurobiological effects of addictive drugs have been
studied in numerous model systems, recently including Drosophila. Some aspects of drug abuse can be readily modeled in
non-human organism, whereas others are much harder to
assess (30). The behavioral and neurobiological consequences
of acute and chronic exposure can be studied relatively easily
and genetic analysis so far shows tantalizing similarities with
some of the findings in mammalian model systems. Addiction
per se, however, and its behavioral correlates such as craving,
withdrawal, drug seeking and continued use despite physical
harm are much harder to study in model organisms, notably
the fruit fly. The work on drugs of abuse in Drosophila has
so far mostly focused on ethanol, cocaine and nicotine.
Flies show a biphasic response to acute ethanol exposure.
After an initial increase in locomotor activity, they become
uncoordinated and eventually pass out. This response is dosedependent and is similar to the response seen in rodents and
humans. Concentrations that lead to increased locomotion in
flies and rats lead to disinhibition and euphoria in humans.
Concentrations that are sedative to flies are also sedative to
humans (31). Genetic screens for altered ethanol sensitivity
have implicated cAMP pathways in acute and chronic ethanol
responses. The first fly mutant with an increased sensitivity to
alcohol was named cheapdate and turned out to be an allele of
the learning mutant amnesiac, encoding a pituitary adenylyl
cyclase activating peptide (PACAP)-like neuropeptide with
homology to the mammalian PACAP (31,32). This complex
role of cAMP signaling in ethanol response has also been
documented in rodents.
As mentioned earlier the addictive properties of ethanol are
more difficult to evaluate, although some interesting findings
have been made in this area. Acute ethanol exposure leads
to a release of dopamine in rodents and blocking this release
blocks the locomotor effect as well as the rewarding properties
of ethanol. In flies a similar reduction in locomotor activity
levels has been observed, although a connection to reward
cannot be made (31). Just as in humans, flies develop tolerance
for ethanol in that repeated exposures delay the effects of
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Figure 3. (A) Coalesced vacuolar structure in spongecake neurodegenerative mutant. (B) A similar membrane-bound vacuole in human brain neuropil associated
with Creutzfeldt–Jakob disease, a prion disease (scale bars ¼ 2 mm). (Adapted from 45 with the kind permission of Elsevier.)
impairment. Flies mutant for tyramine-b-hydroxylase, the
enzyme that regulates octopamine synthesis, have lower octopamine levels and have a reduced tolerance to ethanol. This
finding parallels the fact that mice require an intact noradrenergic system for ethanol tolerance as octopamine is thought to
be functionally equivalent to vertebrate noradrenaline. Preference for ethanol in Drosophila seems closely related to their
levels of alcohol dehydrogenase (Adh), and preference in
adults increases after previous exposure, suggesting a parallel
to the rewarding properties of ethanol in humans (31).
Acute exposure to volatilized cocaine generates dosedependent behavioral responses in flies that are similar to
those observed in rodents. Low doses lead to excessive grooming, moderate doses cause rapid rotation and sideways and
backward walking and high doses cause tremor and paralysis
(33). Molecularly, cocaine has been shown to affect the
re-uptake transporters for catecholamines, so it is not surprising that the behavioral cocaine effects are diminished when
dopamine levels are acutely reduced when flies are fed dopamine synthesis blockers. When dopaminergic and serotonergic
neurons are blocked chronically throughout development, flies
are more sensitive to the effects of cocaine, presumably
because of compensatory adaptations that hypersensitize the
response to dopamine (31).
One property of cocaine response that relates to its addictive
properties is behavioral sensitization or reverse tolerance.
Repeated exposure to cocaine results in an increased sensitivity to the drug and this effect is long lasting. Flies with
reduced tyramine levels do not sensitize, an effect that can
be reversed by feeding flies tyramine. The enzyme that is
required for tyramine synthesis through tyrosine decarboxylation, tyrosine decarboxylase (TDC), is upregulated in sensitized flies and the kinetics of this enzymatic induction
parallel the behavioral sensitization kinetics (34). Suprisingly,
the circadian gene period is required for sensitization, even
though it is not a circadian behavior (35). Mutants with
reduced cAMP-stimulated PKA activity are mostly resistant
to acute cocaine effects and sensitization. Moreover, sensitization and cocaine sensitivity could be altered by changing the
activity in the dopaminergic cells using stimulatory or inhibitory Ga subunits (36).
Nicotine has been less studied in flies, though cholinergic
transmission and the receptors, enzymes and transporter
necessary for it have been identified (37). Flies exposed to
volatilized nicotine are hyperactive at low doses and hypoactive to immobile at high doses similar to the effects described
in rodents. Again, dopaminergic systems seem to be involved
as dopamine synthesis blockers reduce nicotine-induced locomotion (31).
Potential value of fly models for addiction
Flies clearly have at least some of the neuronal machinery
acted upon by substances of abuse such as ethanol, cocaine
and nicotine, and show some of the same ability to modify
their responses to these agents. To this extent, they offer
useful biological models for understanding the mechanisms
of action of the drugs. The missing phenotype, however, to
make them full-fledged models for human addiction is addiction itself. This disparity may highlight interesting aspects of
the evolution of dopaminergic and cholinergic systems.
NATURAL VARIATION
Traditionally, there have been two separate approaches to
behavior genetics in model organisms: the isolation and
manipulation of newly induced mutations (see earlier) and
the measurement and manipulation of naturally occurring
variation in laboratory or wild-caught strains. The latter
assays the extent of relatively mild mutational variation that
survives in the world and exerts effects on behavior. The molecular strategies that have made advances in these efforts possible in humans have also been applied to model organisms.
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The resulting findings live up to the name ‘model’ inasmuch
as they provide a detailed picture of what kinds of variation
exist and what kinds of hurdles must be gotten over to
obtain specific information on relevant genes. They also
provide an interesting assay as to the overlap between those
genes identified in mutant studies and those that vary naturally, as they affect a particular behavioral phenotype.
One such case comes from a study of naturally occurring
variation in the courtship song in Drosophila. This has more
relevance to the foregoing discussion than is immediately
obvious, due to the effect of the circadian period gene on a
critical rhythmic parameter of the song (38). A survey of
such variation and a QTL mapping analysis of recombinant
lines from two variant strains identified three major QTLs,
none of which coincided with any of the genes known from
mutant studies of courtship song in the fly (39). These
results underline the wide-ranging nature of the genes
involved in any behavior. Many genes can affect a given behavior (discussed in 40).
Another approach to the problem of identifying multi-genic
traits has come with the advent of DNA microarray technology. A new approach to this analysis became possible,
based on direct measurement of differences in transcriptional
levels between divergent strains. This was first applied to
strains in Drosophila selected for differences in gravity
response (41). While not all such mRNA expression differences would be due to actual genetic polymorphisms in the
affected genes, some would, and others could be indicative
of relevant phenotypic effects by the polymorphic genes.
Approximately 250 out of 8800 genes were differentially
expressed between the two strains in RNA extracted from
fly heads (42). Functional significance for several of these
genes was verified by testing extant, more severe mutants in
gravity response. In three cases, the predicted direction for
a mutant corresponded to the same direction as the selected
line with the lower expression level of that gene (42). Thus,
for several of the loci, a severe, single-gene lesion could
mimic the selected phenotype that was on the basis of a conglomerate of many genes. In no case, however, was a singlegene effect as strong as that of a selected line.
Both of these examples highlight the multi-genic nature of
natural variants, as well as the extent to which apparently
distant genes can influence behavior.
IMPLICATIONS FOR HUMANS
The foregoing discussion briefly explores the relevance to
humans of gene/behavior studies in the fruit fly. The main
power of the fly models derives from the high level of conservation of function found for many of the relevant genes (e.g.,
period for circadian rhythms, Creb for long-term memory),
and also for the high level of phenomenological conservation
in phenotype for the behaviors they influence. The main caveat
derives from the synthetic nature of some of the models, and
the attendant problem of having taken the human gene out
of context. The approach will validate itself to the extent
that the findings from such synthetic studies show transferability back to the human context.
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