Physiol Rev
83: 687–729, 2003; 10.1152/physrev.00035.2002.
Integrative Physiology and Functional Genomics
of Epithelial Function in a Genetic Model Organism
JULIAN A. T. DOW AND SHIREEN A. DAVIES
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow,
Glasgow, United Kingdom
I. What Is Integrative Physiology?
II. Which Transgenic Model?
A. Choosing your model organism is a trade-off
B. Real physiology in model organisms
III. What Is Functional Genomics?
IV. Why Genome Projects Need Model Systems
V. What Is Reverse Genetics?
A. Reverse genetics demands a genetic model
B. Even human genomics requires simple models
C. Other routes to functional analysis
VI. The “Phenotype Gap”
VII. The Drosophila melanogaster Malpighian Tubule
A. History of Malpighian tubule physiology
B. How is the Drosophila Malpighian tubule organized?
VIII. Physiology and Molecular Genetics of Ion Transport in Renal Function: V-ATPases
A. Drosophila vha55 is single copy and located at 87C
B. The 87C region has been subjected to intense genetic analysis
C. P-element LacZ reporter reveals V-ATPase expression patterns
D. Correlating functional and genetic maps: V-ATPase expression levels
E. V-ATPases are often found in specialized cell types
F. The SzA locus provides multiple alleles of vha55
G. A human renal and auditory phenotype is associated with a plasma-membrane V-ATPase
IX. How Do Chloride and Water Cross the Tubules?
A. Leucokinins selectively increase chloride conductance
B. Patch clamp reveals maxi-chloride channels in tubules
C. Self-referencing electrode analysis shows that chloride moves through stellate cells
D. Tubules contain CLC chloride channels
E. Bartter syndrome type III
F. How does water cross the tubule?
G. Cation and anion transport are spatially separated
X. Pharmacology and Cell Signaling Mechanisms
A. Cell-specific calcium signaling
B. Neuropeptide-stimulated calcium signaling
C. Calcium channels
D. NO/cGMP signaling
E. Cross-talk between NO/cGMP and calcium signaling
F. New targets for drug discovery
G. Insect tubules as targets for selective insecticides
XI. In Silico Mapping of Metabolic Pathways
A. Human, mouse, and gout
B. Drosophila, mice, humans, and gout
C. Disorders of purine metabolism
D. Does Drosophila have a xanthine oxidase mutant?
E. Virtual metabolomics: an index of possibilities
F. Eye color mutants have interesting causes
XII. Drosophila as a Model for Human Disease
A. Can Drosophila cure human disease?
B. How different can you be and still be the same?
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C. Functional genomics offers a future for comparative physiology
D. Functional genomics and integrative physiology are linked
E. A plan for physiologists
XIII. Future Prospects
XIV. Conclusions
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Dow, Julian A. T, and Shireen A. Davies. Integrative Physiology and Functional Genomics of Epithelial Function
in a Genetic Model Organism. Physiol Rev 83: 687–729, 2003; 10.1152/physrev.00035.2002.—Classically, biologists try
to understand their complex systems by simplifying them to a level where the problem is tractable, typically moving
from whole animal and organ-level biology to the immensely powerful “cellular” and “molecular” approaches.
However, the limitations of this reductionist approach are becoming apparent, leading to calls for a new, “integrative” physiology. Rather than use the term as a rallying cry for classical organismal physiology, we have defined it
as the study of how gene products integrate into the function of whole tissues and intact organisms. From this
viewpoint, the convergence between integrative physiology and functional genomics becomes clear; both seek to
understand gene function in an organismal context, and both draw heavily on transgenics and genetics in genetic
models to achieve their goal. This convergence between historically divergent fields provides powerful leverage to
those physiologists who can phrase their research questions in a particular way. In particular, the use of appropriate
genetic model organisms provides a wealth of technologies (of which microarrays and knock-outs are but two) that
allow a new precision in physiological analysis. We illustrate this approach with an epithelial model system, the
Malpighian (renal) tubule of Drosophila melanogaster. With the use of the beautiful genetic tools and extensive
genomic resources characteristic of this genetic model, it has been possible to gain unique insights into the structure,
function, and control of epithelia.
I. WHAT IS INTEGRATIVE PHYSIOLOGY?
Physiology, like most life sciences, is analytical; that
is, a seemingly intractable problem (how an animal
works) is broken down into progressively simpler subproblems. Typically, this means moving from whole animal to isolated tissue (classical physiology). After this,
one of two experimental paradigms is usually employed:
cellular physiology, in which a cell line is derived from the
tissue, in the hope that it expresses at least some differentiated property of interest; and molecular physiology, in
which a gene is cloned, then heterologously expressed in
an experimentally innocuous background (like the Xeno-
pus oocyte) so that, with luck, there are no complicating
factors (Fig. 1).
These are enormously powerful techniques. However, there is a set of problems that these techniques
cannot address, simply because they have taken the system under study away from the physiological context in
which the question was first posed. For example, in the
context of epithelial biology, how does an epithelium
develop? How is the epithelium polarized so that different
transport proteins are found on opposite sides? How are
heterogeneous cell types specified in the tissue, and how
do they interact? What signaling processes allow the tissue to coordinate its function?
FIG.
biology.
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1. Analytical versus integrative
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http://web.bham.ac.uk/bcm4ght6/res.html
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
OO
3
Yeast (Saccharomyces
cerevisiae)
Bug (Escherichia coli)
Rat (Rattus rattus)
Mouse (Mus musculus)
Zebrafish (Danio rerio)
Fly (Drosophila
melanogaster)
Worm (Caenorhabditis
elegans)
4
OO
Human (Homo sapiens)
Organism
Yes
http://flybase.bio.indiana.edu/
http://www.sanger.ac.uk/Projects/C_elegans/
http://genome.wustl.edu/projects/celegans/
http://genome-www.stanford.edu/Saccharomyces/
Yes
Yes
Yes
Yes
Yes
Some
Yes
Yes
Yes
Very hard
Very hard
Yes
Yes
Yes
Yes
No
(Yes)
Partial
Partial
Partial
Yes
Some
Some
Some
Yes
No!
No!
Yes
Classical
Genetics
Targeted
Mutagenesis
Sequenced
Genome
Genetic
Power
Biomedical
Relevance
1. Qualities of major genetic model organisms
TABLE
There are fairly few organisms in which transgenesis
is routine (Table 1). These tend to be termed “genetic
model organisms,” and in addition to transgenesis, there
are frequently genome project resources associated. For
biomedical research, it might seem that only the mouse is
a realistic candidate for transgenics. It is a vertebrate and
has a recognizable body plan, and reasonably well conserved physiology, compared with humans. However, its
high biomedical relevance is offset by its very low genetic
power. Although targeted mutagenesis by homologous
recombination is possible, it is slow and very expensive,
taking perhaps as much as 3 years. This makes it hard to
both generate and analyze a transgenic line in a single
grant cycle.
Although the mouse is, rightly, the biomedical model
of choice, its genetic limitations are manifest. In contrast,
Drosophila represents an ideal trade-off between human
biomedical relevance and genetic power. It is no coincidence that Celera chose Drosophila as the warm-up genome to practice their techniques for humans. In fact,
even simpler models have their place; just as nearly all we
know about developmental genes was first established in
Drosophila, so the bulk of our knowledge about the cell
cycle was derived from yeast, and our understanding of
DNA replication and gene regulation was pioneered by
studies in the humble bacterium Escherichia coli. The
relative merits of model organisms are nicely reviewed
elsewhere (17). It is thus clear that good science is facilitated by a wise choice of model organism that is relevant
to the question being posed, and a thrust of this article
will be that the mouse is not necessarily the automatic
choice for postgenomic physiology.
What is meant by genetic power? Targeted mutagenesis is certainly part of the equation, but there are other
aspects, such as the facility with which forward genetics
can be undertaken. Although mouse is the smallest and
Genetic
Screens
A. Choosing Your Model Organism Is a Trade-off
No!
Mutant
Stock
Centers
II. WHICH TRANSGENIC MODEL?
Serendipitous
Website/Database
The realization that analytical techniques, and in particular molecular biology, cannot give all the answers has
led to a call for a new, “integrative” physiology that seeks
to move from single molecule back to the whole organism
(164). However, rather than throw the baby out with the
bathwater, modern genetics provides tools that let us
combine the strengths of the reductionist approach with
the relevance of organism-level studies. Ideally, we would
like to be able to manipulate specific genes in defined
cells in particular tissues at specific life stages in the
intact organism, as easily as cells can be transfected with
DNA in vitro. Once the problem is phrased in this way, it
is clear that a transgenic approach is needed.
http://www.ncbi.nlm.nih.gov/genome/guide/human/
http://www.ensembl.org/Homo_sapiens/
http://rgd.mcw.edu/
http://www.informatics.jax.org/
http://zfish.wustl.edu/
http://www.fruitfly.org/
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
cheapest vertebrate to keep, the costs are far too high,
and life cycle far too long, to imagine setting up genetic
screens routinely. However, the pay-offs can be large. For
example, an industrial/academic collaboration has produced a systematic ENU mutagenesis of mouse, with a
view to uncovering genes involved in human neurological
deficits (6, 16, 108, 117, 167, 213). An alternative approach
is to draw on the many strains of laboratory rat that have
defined vascular defects and to map (painstakingly) a
variety of blood pressure, osmoregulatory, and renal phenotypes through huge back-crosses, to identify loci by a
quantitative trait locus (QTL) approach (113, 230). It is
highly likely that loci identified in hypertensive rats will
be directly relevant to our understanding of hypertensive
disease in humans. However, these high-profile exceptions tend to prove the rule that rodent forward genetics
is so expensive and time-consuming that it can only exceptionally be undertaken. Should this lack of genetics be
of concern to a physiologist? Perhaps surprisingly, there
are whole swathes of physiological endeavor with roots in
forward genetic screens of “simple” model organisms.
The next section reviews some areas in which our understanding of basic physiology has been advanced by classical genetic studies in Drosophila melanogaster.
B. Real Physiology in Model Organisms
1. Shaker
In the mid 1980s, the first voltage-gated sodium channels were cloned contemporaneously from rat (165), fly
(200), and eel (166). There was massive excitement that
all known ion channels matched the same structural template, with a six transmembrane motif repeated four
times, presumably by two ancient gene duplication events
(202). This argument was dramatically vindicated by the
discovery of an “ancestral,” six-transmembrane potassium channel in Drosophila (41). The route to this discovery was unconventional: whereas the original sodium
channels had been cloned by molecular biochemical
means (cDNA libraries were screened with degenerate
probes derived from peptide microsequence of sodium
channels purified by tetrodotoxin affinity), the Shaker
channel was identified by forward genetics.
A serendipitous behavioral mutation, in which flies’
legs shook under ether anesthesia (41), was analyzed
physiologically and shown to be caused by an underactive
A-type potassium current in muscle (201). The locus was
identified by positional cloning (118) and shown to encode a quarter-sized channel, with six transmembrane
domains. The identity of the locus was confirmed by
showing that the gene was indeed mutated in Shaker
mutants. Although the naı̈ve interpretation would have
been that flies, being primitive, carried an ancestral channel, this would have been far from the truth: Shaker
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homologs were rapidly identified in mammals (199). The
beneficial interplay between physiology and genetics is
nicely illustrated again; although Shaker encoded a potassium channel, A-type potassium currents remained in
Drosophila Shaker mutants, suggesting that further related genes might exist (39). This led to a conventional
library-screening search for related genes, identifying prototypes of three further Shaker subfamilies: Shal, Shab,
and Shaw (39). These genes, in turn, were shown to have
human counterparts (199).
In this case, there is no doubt that the Sh, Shab, Shal,
and Shaw family of channels would have been discovered
in time. However, the use of a genetic model organism
advanced the field by at least a decade. Genetics and
physiology can thus be potent fellows (202).
2. Learning and memory
To most scientists, the conspicuous success of comprehensive forward screens in Drosophila has been the
revolution in our understanding of developmental biology, in recognition of which Edward B. Lewis, Christiane
Nusslein-Volhard, and Eric F. Wieschaus were awarded
the 1995 Nobel Prize in Medicine. However, there are rare
cases where elegant forward genetic screens have advanced our understanding of physiology. Here are two
examples: learning and memory as well as circadian
rhythms.
Although Drosophila is a relatively simple organism
(104 neurons vs. 1012 in humans), it displays recognizable
behaviors, such as learning and memory. With a foresight
that rivaled the great developmental screens, a systematic
olfactory learning screen was employed to demonstrate a
role for cAMP in learning and memory.
The original screen, much copied since, identified a
mutation in a locus (dunce) that was defective in a range
of simple associative learning tasks, but essentially normal in other aspects of behavior (74). Parallel work on
adenosine metabolism showed that dunce mutants had
lower levels of cAMP phosphodiesterase (40, 45). Dunce
was subsequently found to encode a cAMP phosphodiesterase that was expressed predominantly in those brain
regions thought to encompass learning behavior (163).
Support for cAMP as a major messenger in learning and
memory was gained from the discovery that rutabaga,
another learning mutant, showed aberrant adenylate cyclase activity (75), and the gene was subsequently shown
to encode an adenylate cyclase (137).
In parallel with the Drosophila work, cAMP was
shown to play a role in a simple habituation response, the
gill withdrawal reflex, in the sea hare Aplysia (119). The
Aplysia result both extended the phylogenetic scope of
cAMP as a mediator in learning and memory and nicely
illustrates the limitations of work in a nongenetic model,
as it was hard to take the work forward rapidly in Aply-
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sia. In Drosophila, it was possible to extend the results of
the learning screen by crossing dunce to rutabaga and
showing that in the double mutant, both learning behavior
and overall cAMP levels were almost normal (78). Thus a
defect in adenylate cyclase could be compensated for by
a defect in the cognate phosphodiesterase. The principle
of investigating genetic interactions has been extended in
subsequent work. Another feature of a genetic model
organism is the availability of transgenics, and this has
also been brought to bear. Overexpression of a dominant
negative pseudosubstrate inhibitor of cAMP-dependent
protein kinase (cAK) has been shown to disrupt learning,
implicating the kinase in the learning and memory pathways (71). In Drosophila, it is relatively easy to target
expression of transgenes, and this has allowed further
physiological analysis: by overexpression of wild-type rutabaga adenylate cyclase in different brain regions, it was
possible to show that the critical regions for learning and
memory were the ventral ganglion, antennal lobes, and
median bundle (255). It is hard to imagine how such
functional insights could be obtained by other means.
3. Circadian rhythms
Another major screen for genes underlying basic behavior was that for mutants in circadian clock function.
The archetypal clock gene, period, was first found in
Drosophila (131). Although the structure of its encoded
peptide was novel, it is the prototype of a large family
since implicated in circadian function in mammals, and
even humans (224).
The essence of the method is the design of the
screen: only with a robust paradigm can a graduate student be expected to screen the 100,000 flies that might
prove necessary to identify likely mutants. (The scale
required also neatly illustrates the unsuitability of mouse
for such screens.) In this case of Drosophila clock genes,
the screen was suggested by a long history of classical
work on molting and clock genes in insects; adult flies
tend to emerge from the pupa (eclose) around subjective
dawn (49). Therefore, if larvae are reared under a specific
photoperiod then transferred to constant conditions,
those with aberrant clocks will tend to emerge at unusual
times. An experiment like this can be performed on a
large scale with ease, using automated collection equipment (49): flies selected by the screen are collected and
bred, and a small fraction of them can be expected to
have a definable genetic lesion in a clock gene. This
screen, or similar, was used to identify many of the major
components of the circadian clock, for example, period
(131) and timeless (207).
There is another lesson to be learned from this example. Although there is a massive literature on circadian
clocks in humans, the human period-1 gene, on chromosome 17, was identified only in 1997 (220, 224), 10 years
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after the Drosophila sequence had been published (48).
So, progress in human physiology might sometimes be
accelerated by close attention to progress in simpler models.
4. The inositol trisphosphate receptor
Calcium signaling is a fundamental process, and in
most cells, calcium is released from internal stores in the
endoplasmic reticulum (ER) in response to hormone signaling, through the action of inositol 1,4,5-trisphosphate
(IP3) on its receptor (IP3R), a large ER calcium release
channel (24, 25). While most studies of calcium signaling
have been performed on cultured cells, because of the
experimental tractability, there have been some genetic
experiments. In mouse, both the natural opisthotonos
mutant and targeted knock-outs of the IP3R show multiple
neurological defects and die soon after birth, confirming
that, despite the existence of alternative ER release channels, the IP3R is essential for life. In Drosophila, the lethal
phenotype is reproduced; however, here it has been possible to produce a range of alleles of differing severity (an
“allelic series”), that includes severe hypomorphs, and
lethal alleles that make viable, but hypomorphic, transheterozygotes. It is also relatively straightforward to construct chimeras so that lethal mutations can be studied in
clones of homozygous cells in an otherwise viable heterozygote. In this way, slightly more informative results can
be obtained; the key defect in hypomorphic alleles seems
to be delayed larval development and adult emergence
(101, 233). This implies a critical role for IP3R in the
endocrine control of growth and development, probably
connected to the release of the molting hormone ecdysone (233, 234). Intriguingly, although IP3R is single copy
in the Drosophila genome, it does not seem to be necessary for visual phototransduction, a classical phospholipase C-mediated pathway (2, 182).
Such insights are not confined to studies in Drosophila. Similar analysis of IP3R function in C. elegans was
taken in a slightly different direction by the generation of
green fluorescent protein (GFP)-tagged IP3Rs, and by
overexpressing the IP3 binding domain, to produce a dominant negative effect (19). The former technique provides
a dynamic means of following levels and distributions of
gene products in living animals and showed that, although
IP3R is presumably pretty ubiquitously expressed, it is
found preferentially, not just in the nervous system, but in
the pharynx, gonad, intestine, and excretory cell (19).
From here, it was relatively straightforward to map the
gene’s control regions and correlate them with the observed expression levels (90). The latter, dominant negative approach (together with RNA interference) showed a
role for IP3R in two behaviors at opposite ends of the
alimentary canal: feeding and defecation (235). The use of
a heat-shock promoter for the dominant negative con-
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
struct allowed intervention in the adult, avoiding the confounding effects of disrupting IP3R during development.
These interventions would be difficult in a larger animal.
It has also been shown in a yeast two-hybrid screen that
IP3R interacts directly with myosin; the availability of a
simple model allowed these results to be tested directly in
C. elegans, confirming the functional significance of the
interaction in vivo (236).
From the examples above, it can be seen that harnessing the power of even a simple genetic model can
produce fundamental insights in mainstream (i.e., de
facto mammalian) physiology. It is of interest to note,
though, that the three examples shown above are all of
the “brain and behavior” phenotypic class. There have
been very few successful attempts to exploit Drosophila
or other simple models in other areas of physiology. Later,
this article will address perhaps the most developed such
phenotype for epithelial function, ion transport, and cell
signaling, the Drosophila Malpighian (renal) tubule. This
will illustrate both that real physiology is possible and
that it can provide useful general insights.
III. WHAT IS FUNCTIONAL GENOMICS?
“Functional genomics” is a fashionable bandwagon,
which has been hijacked to a number of laudable purposes. Originally, it was seen as a (necessarily) highthroughput route to assigning functions to new genes. As
a first step into the postgenomics era of functional genomics, a range of ingenious, large-scale “chip” technologies
have been devised that offer the promise of identifying
candidate genes based on their differential expression in
two tissues or in disease states. This is extremely attractive, particularly to the drug industry, because it allows
the rapid identification and patenting of interesting genes
without necessarily knowing their function (204). It is
now becoming increasingly clear that, although highthroughput technologies provide valuable lists of candidate genes, they can provide only caricatures of function.
Ultimately, then, it is necessary to roll up one’s sleeves and
work up the function of individual genes and gene families.
This activity is as deserving of the epithet functional genomics as any high-throughput technology. It is thus perhaps
useful to consider functional genomics as any elucidation of
function in the context of an organism’s genome. In the next
section, we will make the case that this functional genomics
requires exactly the same approaches and reverse genetic
technologies as integrative physiology, to the extent that the
two areas can be considered to have merged.
human genome has been sequenced completely, the nucleotide sequence of every gene involved in any disease
process must have been deduced. Unfortunately, connection between the two is not automatic. Even after coding
sequences have been identified in humans, it remains to
correlate each of them to their physiological function.
There are essentially three ways in which this will be
possible (Fig. 2).
1) A substantial fraction of genes will already be
known, and their physiological roles will be effectively
elucidated. This will include a number of the genes responsible for major genetic diseases, which are being
directly sought in parallel with the main human genome
project.
2) For a significant further fraction of the remaining
genes, it will be possible to infer function either by similarity to other known human genes or to well-known
genes characterized in other organisms. Additional information will become available from expression patterns or
from the chromosomal localization of the gene mapping
close to a known and plausible genetic disease.
3) However, a significant fraction of genes (from
most genome projects, estimates are around one-third)
will be genuinely new, with no structural similarities that
could suggest likely avenues of research. It is for this
class of gene that reverse genetics will be particularly
important.
V. WHAT IS REVERSE GENETICS?
Reverse genetics is the deduction of gene function by
analysis of corresponding mutant phenotypes (139, 176,
196, 197). This is acknowledged as the quickest and most
promising way of inferring function for a new gene (Fig.
3). It relies on the presence of a homologous (or at least
analogous) gene, in a model organism in which a gene
knock-out can be created (135, 141, 179). The success of
reverse genetics at the genome project level thus depends
on the range of model organisms available; a wider range
maximizes the probability that a homologous gene can be
identified.
IV. WHY GENOME PROJECTS NEED
MODEL SYSTEMS
At first, it might seem self-evident that genome
projects are a good thing. In particular, now that the
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FIG.
3. Reverse genetics.
Perhaps less obviously, it further relies on the availability of a phenotype that can be studied for effects of the
induced mutation. In other words, there is no point in
mutagenizing a gene in a model organism in which there
is no assay for effects of the mutation! For example, it
would be a waste of effort to mutagenize a neural-specific
gene in an organism too small to allow neurophysiological
analysis. The choice of model is thus a trade-off between
biomedical relevance, genetic power, and amenability to
physiological analysis, exactly as we have outlined for
integrative physiology above.
693
also essential. This explains the apparent contradiction
that human genomics in particular, and biomedical science in general, has hugely cross-subsidized fundamental
research in genetic models. However, as described in
Table 1, the choice of transgenic model is not automatic.
There is a trade-off between biomedical relevance (with
humans top, and mammals next), cost, and convenience
of life cycle (which favors smaller model organisms),
ethical desirability (which favors smaller model organisms), and power of the genetic tools available (which
favors smaller model organisms).
At first, mouse would seem to be ideal, as it is the
only mammalian model for which detailed genetic intervention is routine. It is possible to inactivate genes with
single base-pair precision by homologous recombination
and to introduce transgenes. However, gene knock-outs
are often lethal sufficiently early in development as to be
uninformative. In mouse, the ability to introduce more
subtle defects (for example, to inactivate gene function
only in adults, or only in a particular tissue) is highly
limited and must be set up on a case-by-case basis, compared with other models, like Drosophila or C. elegans,
where generic technologies are available. Accordingly, for
the foreseeable future, these organisms are likely to be
ideal models for integrative physiology.
C. Other Routes to Functional Analysis
Reverse genetics demands at the least some means of
targeted mutagenesis of a gene of interest. Although generic approaches are being developed [RNAi (109) is an
example], targeted mutagenesis has generally only been
developed in organisms in which there is a considerable
volume of classical genetics. Typically, this means that there
is also a large body of literature covering known genes,
visible markers, and recombination or physical maps. Such
organisms have also invariably been selected as early subjects for genome projects. This confluence of classical and
reverse genetics, together with genomics, provides a working definition of “genetic model” organism. Such organisms
form a short but distinguished list and have impacted on
nearly every field of biology to date, except physiology.
Although it is generally accepted that reverse genetics is the most powerful method of elucidating function of
novel genes, there are other special cases that are nonetheless worth noting. For example, cluster analysis of
microarray data tends to gather together functionally related genes. So if a single novel gene clusters with known
genes that are all implicated in a single process (like
amino acid metabolism), then a clear functional hypothesis presents itself (76). Similar routes can be plotted at
the proteome level, although there are more potent ways
of identifying functional complexes. By systematically
fishing out the partners of epitope-tagged proteins in yeast, it
has proven possible to identify the constituents of multiple
hetero-multimeric complexes (86, 105). Similarly, more advanced computational techniques may allow hypothetical
proteins to be classified based on structural similarities
more subtle than conventional BLAST searching at present
(77, 111, 175). However, these exceptions also illustrate the
rule: they all generate hypotheses that must be tested experimentally. So reverse genetics is still a required part of the
transition from gene to function, and physiologists are essential participants in the endeavor.
B. Even Human Genomics Requires Simple Models
VI. THE “PHENOTYPE GAP”
Because reverse genetics will be essential to human
functional genomics, it follows that model organisms are
Unfortunately, exactly the qualities that make for a
good genetic model militate against physiology. For rea-
A. Reverse Genetics Demands a Genetic Model
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
sonable genetics, small size, low cost, rapid life cycle
organisms are needed. This means that historically there
is little depth of physiology available. This mismatch has
been recognized by the genome project community and
has been termed the “phenotype gap” (37, 38).
It is easy to demonstrate the phenotype gap objectively. For example, most rodent physiologists have concentrated on rats, guinea pigs, and rabbits rather than the
smaller genetic model, the mouse. This difference can be
marked: a literature search of Science Citation Index
publications shows that for almost any keyword or tissue
of choice, the recent literature on rats is about fivefold
larger than on mouse (Table 2).
Although rat genetics and genomics will eventually
rival those of the mouse in power, there is a pressing need
to translate rat physiological methodology into mouse,
and so provide the widest range of physiological assays
possible. This will maximize the chances that mutation of
any particular gene will have a detectable and informative
physiological phenotype.
Genome projects acknowledge the need for physiologists to help close this phenotype gap, and this provides
some of the most exciting openings for physiologists into
the 21st century. Exactly how to ride this wave is discussed later.
In the next section, we will give an example that
draws together the strands of the article: that combining
physiology and genetics in a simple genetic model provides an ideal system for truly integrative physiology, and
that by doing so physiologists can contribute to postgenomics by closing the phenotype gap.
VII. THE DROSOPHILA MELANOGASTER
MALPIGHIAN TUBULE
Insect Malpighian tubules perform excretory and osmoregulatory roles analogous to vertebrate renal tubules and
have been studied extensively in a literature that dates back
over half a century. However, most studies have focused on
TABLE
2. Demonstration of the phenotype gap
Keyword
By tissue
Brain
Kidney
By target
Kinase
Calcium
By concept
Transport
Signal
Rat
Mouse
36,279
8,138
6,730
2,351
11,060
12,282
4,222
2,356
9,246
10,996
1,786
5,276
Comparison of the number of papers published (BIDS ISI title/
keyword/abstract search 1995-8) on transport and cell signaling using a
physiological (rat) and genetic (mouse) model.
Physiol Rev • VOL
far larger insects. Drosophila tubules are among the smallest ever studied, measuring ⬃2 mm long by 35 ␮m in diameter, and are comprised of ⬃150 cells. We have recently
found that the Drosophila renal tubule can be used for
physiological studies of fluid secretion (69) and that it shows
a rich pharmacological repertoire that includes the cAMP,
nitric oxide (NO)/cGMP, and Ca2⫹ signaling pathways (63,
66). This means that, for a very wide range of developmental, transport, and signaling genes, the tubule is probably the
most robust Drosophila phenotype presently available.
A. History of Malpighian Tubule Physiology
This has been reviewed briefly elsewhere (63). Marcelo Malpighi (1628 –1694), physician to the Pope, is well
known as an early adopter of the microscope, contemporaneously with Hooke, and followed the discoveries of the
great William Harvey on the nature of circulation. However, he was a comparative anatomist and also turned his
microscope to insects, discovering the eponymous insect
Malpighian (renal) tubule. However, it was not until the
20th century that insect physiology became feasible.
Wigglesworth, then his pupil Ramsay (183), was able to
demonstrate that insect tubules indeed produced urine,
and Maddrell (150) was able to demonstrate endocrine
control, using one of the more remarkable epithelial
model systems, the tubule of the bloodsucking bug Rhodnius prolixus. As this bug feeds on huge blood meals
perhaps once in 6 mo, and must then fit into cracks in
walls and floorboards to escape discovery, it has a potent
control of its diuresis and can accelerate urine production
by more than a 1,000-fold after feeding (153). Since then,
there has been a large literature on insect tubules from
many species (for reviews, see Refs. 50, 62, 63, 66, 150,
152, 153, 173, 177, 231), and it is accepted that the insect
tubule is both a useful epithelial system and a valid target
for insecticide development. This classical literature suggested that the Malpighian tubule of Drosophila melanogaster might be amenable to physiological analysis, although it was smaller than any previously studied.
B. How Is the Drosophila Malpighian
Tubule Organized?
1. The classical view
Insect Malpighian tubules are simple epithelia, freefloating in the insect’s hemocoel (there is no vascular
circulatory system beyond a rudimentary tubular heart)
(43, 247). Similarly to birds, and for similar reasons of
water economy, the tubules do not open directly to the
outside, but join to the alimentary canal at the junction of
the endodermal midgut and the ectodermal hindgut (Fig.
4). The relative experimental accessibility of the insect
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695
phology, of the tissue of interest. In a nonmodel organism,
our understanding of a tissue is based on the experimental techniques available and the training of the scientist. It
is thus only possible to obtain an experimenter’s view of
the tissue organization. In certain genetic model organisms, in contrast, a technique known as enhancer trapping
can provide an insight into how the organism organizes a
target tissue.
3. The principle of enhancer trapping
FIG. 4. Classical morphology of the adult Drosophila melanogaster
Malpighian tubule. [From Wessing and Eichelberg (242).]
tubule compared with the vertebrate kidney tubule is an
advantage; they are similar in overall dimensions. In the
case of Drosophila, there are four tubules, joined in pairs
through short common ureters to the alimentary canal.
Interestingly, the right-hand pair are longer, with a prominent white initial segment, and are always placed anteriorly in the body cavity. In contrast, the left-hand pair was
thought to lack initial or transitional segments, and always sit posteriorly, within the abdomen (242). (Incidentally, this could provide a useful screen for mutants of
sinistrality, an area hardly explored in fly). There are two
major cell types: the larger type I (or principal) cell and
smaller intercalated type II (or stellate) cell (242).
2. Enhancer trapping as a tool to
understanding organization
A fundamental part of a physiological study is to
analyze the structural organization, or functional morPhysiol Rev • VOL
Enhancer trapping has been widely used in the context of development, for which it was originally developed
(23). It makes use of an engineered transposon, in Drosophila usually the P-element (193). Transposons are a
class of semi-autonomous mobile DNA elements (loosely
similar to retroviruses), found widely in all organisms,
that encode an enzyme (transposase) that can recognize
the stereotyped ends of the transposon, and catalyze its
excision and reinsertion in the genome (187). In most
organisms, this parasitic process is largely benign, although occasionally a cell can be killed or transformed by
disruption of a key gene (“insertional mutagenesis”). In
Drosophila, one transposon (the P-element) has been
engineered to carry a variety of genetic constructs that
permit a remarkable range of genetic manipulations in the
intact organism (192). For enhancer trapping, a P-element
is used in which the transposase gene has been replaced
by three elements: 1) a white marker gene, which gives
red eyes in flies carrying the transposon and so allows it
to be tracked through crossing schemes; 2) a reporter
gene (such as lacZ), coupled to a weak, permissive promoter [expression of the lacZ gene is then sensitive to its
genomic context (i.e., where the transposon is sitting
within the genome, relative to other genes and their natural promoters and enhancers)]; and 3) a linearized plasmid, which allows the site of insertion to be determined
by a technique known as plasmid rescue.
Without a transposase gene, this element is capable
of mobilization but cannot transpose itself. It is thus
trapped within the genome unless provided with a source
of transposase. This enzyme can be supplied by crossing
flies carrying the P-element to another line in which a
P-element has been serendipitously inactivated by an imprecise excision event in the past. In the ⌬2,3 line, a
functional transposase is produced only in the fly’s germ
cells, and as the element is missing one of its ends, it
cannot be mobilized by its own enzyme (187). In an
enhancer trap screen (Fig. 5), flies carrying both the enhancer trap element and a source of transposase are
called jump-starters. In each of their sex cells (usually
sperm: males are used by preference because there is no
male meiotic recombination), there is the potential for the
enhancer trap element to mobilize and reinsert somewhere else in the genome. By segregating and breeding
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
FIG.
5. Enhancer trapping.
true from hundreds or thousands of progeny of such
jump-starter males (and by ensuring that the ⌬2,3 transposase source is quickly crossed out of each line), a range
of new, stable, insertion sites will be obtained. In some of
these, the enhancer detector will have landed near some
unknown gene with patterned expression in the tissue of
interest, so by staining representatives of each line for the
reporter gene, the 10% or so of genes of interest to a
particular experimenter can easily be determined. (Of
course, the remaining lines may be of interest to another
experimenter, and it is common practice to exchange
panels of lines within the Drosophila community.)
This description may sound esoteric in a physiological context, but its execution is straightforward, requiring
only a series of crosses over months, and the physiological payback is huge, because the technique reveals the
organism’s, rather than the experimenter’s, view of the
organization of the tissue. In contrast to the methodology,
the results are visually striking and easily grasped.
Classically, the anterior tubules were considered
to have three domains and the posterior tubules one.
We found lines that reflected these domains of expression, consistent with the previous studies (Fig. 6, A–D);
however, we also found new aspects of organization.
The lines that marked out initial and transitional segments in the anterior tubule also identified miniature
domains in the posterior tubule (Fig. 6, A and B). The
anterior and posterior tubules thus differ quantitatively
in the extent of these regions, rather than qualitatively
in the nature of their specification. The main segment
of the tubule (Fig. 6C) can be divided into two, with a
prominent lower tubule domain (Fig. 6D) that is in turn
separable into three domains (214). Critically, then,
there are six regions in both anterior and posterior
tubules, and several of them had been refractory to
experimental identification in the absence of enhancer
trap studies.
4. Enhancer trapping can detect tubule-specific genes
6. Enhancer trapping reveals multiple cell types
As part of a 1,500-line screen using the P{GATB}
transposon (252), 750 lines were screened for tubulespecific patterns of expression in larvae and adults (214).
Of these, ⬃10% were informative, and of these, ⬃20 lines
were used to delineate boundaries of expression in the
tubule. In most studies, enhancer trapping is used as a
rapid means to identify genes of interest, and so generate
data similar to the results of a differential cDNA library
screen. However, the patterns themselves are informative, as they reflect the actions of combinations of cellspecific transcription factors on the enhancer detector. In
this way, they genuinely reveal aspects of the tissue’s
spatial and temporal organization.
The principle of these studies can be extended to
cell types (Fig. 6, E–H). Surprisingly, few lines marked
out all the principal cells in the tubule: the normal
pattern was for only a subset of principal cells to be
marked (Fig. 6E). This is significant because it means
that morphologically indistinguishable cells are expressing different genes, and thus presumably performing different functions.
The other major cell type, the stellate cell, is also
clearly labeled by this technique (Fig. 6F). Interestingly,
the two lines that marked stellate cells had the most
restricted pattern of expression in the rest of the fly of any
studied. This may imply that their function is unique and
Physiol Rev • VOL
5. Enhancer trapping reveals regional specialization
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FIG. 6. Gene expression domains
(A–D) and cell types (E–H) in the Malpighian tubules revealed by enhancer
trapping. A: initial and transitional segments by inclusion. B: initial and transitional segments by exclusion. C: main
segment. D: lower tubule. E: a subset of
principal cells. F: stellate cells; in this
case, lacZ expression is detected with a
fluorescein-tagged antibody, and nuclei
have been counterstained with ethidium
bromide. G: bar-shaped cells, unique to
the initial and transitional segments. H:
myoendocrine cells of lower tubule and
posterior midgut. [From Sozen et al.
(214).]
that they express relatively unusual transcripts. The distribution of stellate cells also helps to cross-validate the
domains of expression outlined earlier: stellate cells are
not found in the lower tubule domain, and their shape
changes from stellate in the main segment to bar-shaped
in the initial and transitional segments (Fig. 6G). The
bar-shaped cells can in turn be distinguished from stellate
cells by a single line that labels only the former.
There are other, minor cell types, such as tracheal
cells, and tiny myoendocrine cells found only in the lower
tubule region (Fig. 6H). Taken together then, there are six
cell types and six regions in this tiny tissue.
Physiol Rev • VOL
7. Genetic domains can be quantified
by counting nuclei
At this stage, it would be traditional to clone the
flanking genomic regions by plasmid rescue and seek to
identify the genes responsible for the patterns of expression that had been observed. However, we tried to quantify the sizes of different domains and to compare the
sizes and stability of domains reported by different lines
to see whether they were reporting the same boundaries.
This would render the description of tubule structure at a
quantitative rather than anecdotal level. In fact, it was
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
straightforward to label tubule nuclei with ethidium bromide and to count the nuclei in each domain (Fig. 7A).
Remarkably, it was possible to show that the positions of cell boundaries, and the numbers of cells in each
region, were fixed to near single-cell precision (Fig. 7B).
This means that every cell in the tubule has a precise view
of its positional identity, and the development of the
tubule is extremely robust and deterministic. Although all
the insertions studied were homozygous viable, some are
nonetheless disruptants of the genes in which they were
inserted (see below). Despite this, cell numbers did not
vary between lines, in any of those we studied, although,
of course, there are some developmental mutants in
which tubule development is perturbed (106). In this simple, one-dimensional system, it may be possible to undertake identified-cell epithelial physiology, for almost the
first time. Although this is already technically possible for
morphologically distinguishable cells (for example, Refs.
30, 95), it is clear that morphologically indistinguishable
cells can express different genes (Fig. 6), and so have
different functions (151, 171, 186). Enhancer trapping,
and particularly the vital labeling of domains of gene
expression, neatly provides a solution to this issue.
8. The GAL4/UAS system
The level of sophistication revealed by enhancer
trapping might seem daunting, but the same enhancer
trap technology provides the tool to intervene genetically
in any population of cells that can be defined by an
enhancer trap line.
In the second generation, or binary, enhancer trap
system (Fig. 8), the reporter gene is the yeast transcription factor GAL4. This appears to be almost perfectly inert
within the Drosophila genome, and so normally has no
effect on the host organism. However, it is capable of
driving transgenes under control of the yeast UASG promoter. Thus, given a panel of informative GAL4 lines, as
described above, any genetic construct of choice (not just
lacZ) can be directed to any of the lines. The GAL4/UAS
system additionally acts as a switch, in that high levels of
driven expression contrast with very low background
levels. The binary nature of the system means that a new
transgenic line can be generated in ⬃3 mo, without having
to repeat the enhancer trap search for GAL4 drivers. This
compares very favorably, in both time and cost, with the
effort to make knock-in constructs for mouse and to
generate and test the transgenics. There is another key
advantage of the system: if a deleterious gene product (for
example, a gene in the apoptosis pathway) is to be expressed, then the UAS stock can be kept safely without
serious loss of fitness. The deleterious construct is only
expressed in the progeny of the cross between the parent
lines.
This GAL4/UAS binary system opens possibilities to
the physiologist that are mouth-watering. At present, it is
a technique specific to Drosophila, and a potent reason
why experimenters should relish, rather than dread, the
opportunity to work in this organism.
9. Correlating functional and genetic maps
FIG. 7. Quantifying enhancer trap domains. A: lower tubule domain
marked with fluorescein-tagged anti-lacZ, nuclei visualized by counterstaining with ethidium bromide. In this case, there are 26 nuclei in the
lower tubule domain. B: summary of domains (right) and the numbers
of principal and stellate cells found in each domain (left). In each case,
the standard error is ⬍1.
Physiol Rev • VOL
Enhancer trapping suggested a clear, reproducible
structure for the Malpighian tubule. However, it is critical
to establish whether this genetic map has any relevance to
physiology or is merely some genetic curiosity. To accomplish this, the genetic map was tested with a battery of
functional assays, and in each case functional and expression domains were found to coincide perfectly. This section reviews evidence for the congruence between genetic and functional maps.
A) FLUID SECRETION. The key property of the Malpighian
tubule is its ability to produce an isotonic fluid. Indeed,
for most insects, this is the only property that has been
measured. It is possible to adopt the classical Ramsay
assay for fluid secretion to this tiny tissue, and the Drosophila tubule is actually very robust ex vivo, maintaining
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699
FIG. 8. Binary enhancer traps. A:
binary enhancer trap, using the GAL4/
UAS system. B: example of binary system, in which the c724 stellate cell-specific GAL4 driver is used to direct green
fluorescent protein expression to stellate
cells under control of UAS. Unlike the
lacZ staining shown earlier, this is a live
tubule.
stable secretion rates of 0. 5–1 nl/min for several hours in
appropriate medium (69). It is possible to map secretion
rates as a function of length along the tubule, although the
spatial resolution of the assay is not as good as that of
enhancer trapping. However, it was possible to show that
the main segment of the tubule secretes fluid, while the
lower tubule reabsorbs it (Fig. 9) (171).
B) ALKALINE PHOSPHATASE. A powerful product of the
enhancer trap technology is the rapid identification of the
genomic location of the insertion and thus frequently the
gene near which the P-element is inserted. The lower
tubule is delineated by two lines, which represent independent insertions in the same gene, encoding an alkaline
phosphatase (Aph4) with greatest similarity to the human
liver/bone/kidney (ALPL) type (253). In humans, ALPL is
an ecto-enzyme that metabolizes phosphoethanolamine
(PEA) and pyridoxal-5’-phosphate (PLP). In Drosophila,
Aph4 is expressed only in the lower domain of the Malpighian tubule, and in a small group of cells, the ellipsoid
body in the brain (253). It also appears to be an ectoPhysiol Rev • VOL
enzyme, as histochemistry for alkaline phosphatase activity labels only the apical surface of the lower tubule.
Remarkably, although the genome project has annotated 13 alkaline phosphatase genes in Drosophila, the
expression patterns of the Aph4 enhancer trap insertions
perfectly match the alkaline phosphatase histochemistry.
This implies either that the other 12 alkaline phosphatase
genes are not expressed significantly in the adult or that
the nitro blue tetrazolium-based histochemical stain does
not detect all the alkaline phosphatase activities in Drosophila.
In vertebrates, the roles of alkaline phosphatase are
not entirely clear. In humans, mutations in ALPL are
associated with hypophosphatasia (241). In mouse, the
homologous tissue nonspecific (TNAP) isoform, when
mutated, causes fatal seizures 2 wk after birth; these can
be survived if pyridoxal is administered. There is also
some evidence of hypomineralization in teeth (240). At
least one human case has been reported with similar
symptoms (244). However, the enigmatic renal role of
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
alkaline phosphatase might be addressed in Drosophila,
as the P-element insertions in Aph4 both disrupt expression and cause a transport phenotype (Fig. 10).
10. Phylogenetic scope of enhancer
trapping technology
FIG. 9. Fluid secretion as a function of length along the Malpighian
tubule. Tubules were isolated under paraffin oil, varying lengths left in
the bathing drop of saline, and droplets of fluid collected from the ureter
at regular intervals. (The nature of the assay makes it hard to obtain
estimates from the extreme tip or close to the ureter of the tubule.)
[From O’Donnell and Maddrell (171).]
It is clear from the above that enhancer trapping
delivers results that are highly relevant to the physiologist
at several levels. First, it provides a screen for tissuespecific genes with added informational content by virtue
of the expression pattern reported. Second, enhancer
traps themselves report pattern in a tissue at a level that
could probably never be deduced by explicit experimentation, so delivering new understanding of tissue organization. Third, second and later generation enhancer traps
provide generic technologies for conditional expression,
overexpression, and gene tagging. Fourth, enhancer trap
insertions have the potential to be mutagenic, either directly by virtue of their insertion site or after imprecise
excision of the P-element. They are thus vital components
of a functional genomics strategy (22).
Obviously, these experiments can never be attempted in humans, so it is necessary for human physiologists to resort to model organisms. Enhancer trapping is
at its most developed in the fly, with multiple classes of
P-elements available, including the second generation
GAL4/UAS binary system, together with several ES and
other gene-trap vectors (22, 189).
In Arabidopsis, enhancer trapping and gene trapping
have both been used, usually with the maize Ds transposon and GUS as the reporter gene (129, 221). However,
FIG. 10. P-element insertions near Aph4 disrupt
alkaline phosphataase expression and reduce fluid secretion in Drosophila tubules. A: histochemistry for
alkaline phosphatase in tubules of wild-type flies,
showing activity in lower tubules. B: homozygous Pelement insertions, showing reversed expression in
main segment, but not lower tubule. C: heterozygous
flies, showing ectopic expression throughout the tubule. D: resting and stimulated (cAMP, 10⫺5 M at 30
min) fluid secretion rates of tubules from wild-type
(white squares) and homozygous P-element insertion
(black diamonds) fly lines. Both basal and stimulated
secretion rates are reduced in homozygotes. Error bars
denote SE, with number of tubules in each treatment in
parentheses. [From Yang et al. (253).]
Physiol Rev • VOL
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INTEGRATIVE PHYSIOLOGY AND FUNCTIONAL GENOMICS
the technology does not appear to have been used to its
full potential (22).
In zebrafish, a Rous-sarcoma virus LacZ reporter injected into embryos showed patterned expression, although in only one line was it heritable (18). This is a
fairly laborious method, as each line is derived from a
separate injected embryo; there is no remobilization of a
preexisting integrated reporter gene.
In C. elegans, transposable elements are not easily
tamed, because of the lack of clean germ line-specific transposase expression (22). However, it is relatively easy to
introduce heterologous DNA into worms, and this has been
used for a form of promoter trapping in which random
genomic fragments are cloned upstream of a LacZ reporter,
then transformed into worms (145). If this technique is
performed systematically on a large enough scale, it can
emulate some of the functionality of enhancer trapping.
Enhancer trapping is technically possible in mouse,
although the large genome size means that fewer insertions are likely to be informative. Additionally, the effort
in generating and maintaining sufficient stocks (the limiting factor in Drosophila screens) makes a rigorous enhancer trap screen unlikely. However, promoter and
gene-trap techniques have been applied widely (191).
Such studies usually rely on a splice acceptor site within
a retrovirus vector, producing a lacZ fusion protein with
the initial exons of the mouse gene. Such insertions are
both rarer and much more mutagenic than enhancer
traps, because they are guaranteed to disrupt the protein.
Rather than try to jump the vector within the mouse,
random insertions are generated within clones of embryonic stem (ES) cells. These can then be grown up into
whole mice as desired. This can produce a valuable, highthroughput screen, as insertions can be screened for expression, or the insertion site sequenced, within ES cells;
transgenic mouse lines are then only generated from cell
lines of interest (116). At one level, this has less appeal
than Drosophila enhancer trapping, because it implies
preselection by the experimenter, and so reintroduces
experimenter bias into the paradigm. However, at another
level, it provides a potentially valuable route to reverse
genetics of defined genes, perhaps those identified from
high-throughput methodologies. Such an approach has
been taken to a highly commercial level, with experimenters able to purchase ES clones with defined insertions
from a bank of over 200,000 frozen lines (254) and produce their own transgenic mice (http://www.lexgen.com/
omnibank/omnibank.htm). There are obvious cost and
intellectual property issues in such an approach.
Elements of the GAL4/UAS system are now used in
mouse, in order specifically to provide switched expression (138). However, in mouse, GAL4 cannot be used as
an enhancer trap element, because of the scale of the
experiments implied. It is expressed downstream of a
specific promoter, which must be characterized painstakPhysiol Rev • VOL
701
ingly first. There is no equivalent for panels of enhancertrap insertions into anonymous genes that can be used for
expression studies.
11. Emulating enhancer trapping in other organisms
Even if the GAL4/UAS system cannot be deployed in
a target species, it would be highly desirable to obtain
insight about the spatial organization of tissues of any
species of interest. Is this achievable without true enhancer trapping? We propose two alternatives for studies
in organisms where enhancer trapping is not feasible:
comparative in situs and random in situs.
For the comparative technique, enhancer trapping of
the analogous tissue in a suitable model will lead to a
number of candidate genes. These can be used as probes
for comparative in situs in the target tissue. For example,
the mosquitoes Anopheles gambiae and Aedes aegypti are
major vectors of disease (with completed genome projects),
but transgenesis is still at too early a stage for routine
enhancer trapping (3). However, some of the genes identified in an enhancer trap screen of midgut in Drosophila
might be expected to delineate patterns in the Anopheles
midgut, and so enhance our understanding of the target
tissue through which parasites must migrate as part of their
maturation. Such cross-species hybridizations are, of
course, technically demanding, so an alternative approach
might be to clone target species homologs of the genes
identified in the genetic model. Another approach would be
to use knowledge of the genes to identify functional assays
(such as histochemistry) that can be used across species.
This approach will be illustrated in section XIIIB1A, using our
knowledge that alkaline phosphatase marks out the lower
tubule domain (see sect. VIIIA9B).
For random in situs, we propose large-scale in situ
hybridization with random cDNAs from the tissue of interest. This approach could go hand-in-hand with an expressed
sequence tag (EST) project, an accepted means of discovery
in a tissue of interest. The power of the approach would be
greatly increased by normalizing the library, a technique that
selectively enriches the abundance of rare transcripts to
nearly the same level as abundant transcripts within the
library (212). The random in situ approach has the advantage, common to classical enhancer trapping, that domains
of functional expression are mapped impartially, without
introducing experimenter bias. Furthermore, if in situs could
be performed systematically in a 96-well format, then relatively few plates could generate, in a few weeks, information
similar to that obtained by our two-year enhancer trap study.
VIII. PHYSIOLOGY AND MOLECULAR GENETICS
OF ION TRANSPORT IN RENAL
FUNCTION: V-ATPASES
A critical test of the enhancer trap model would be to
map the expression of the major transport processes that
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energize fluid secretion, because this is known to be
patterned according to the genetic domain model. In fact,
these studies provided both a validation of the existing
model and some more general insights into the partitioning of transport roles between cell types.
Historically, transport across most insect epithelia
was thought to be energized by a primary, electrogenic,
chemisosmotic K⫹-ATPase (98). The most accessible and
spectacular example of this transport could be found in
the larval lepidopteran midgut, which can develop lumenpositive potentials in excess of 100 mV, drive currents of
over 1 mA/cm2 (96, 98, 100), and in some species generate
a luminal pH in excess of 12, the highest in biology (58,
70). Within the midgut, the apical membrane of a specialized goblet cell was decorated with an array of 10-nm
spheres (5), termed “portasomes” (97), and it was on this
membrane that active K⫹ transport was shown to take
place (67). However, painstaking biochemical purification
of the goblet cell apical membrane of lepidopteran midgut, based on purification of the portasomes (47), identified not a P-type ATPase similar to K⫹-H⫹-ATPases of
vertebrates, but a V-type H⫹-ATPase (205, 246). This was
the first demonstration that V-ATPases, considered important for vacuolar and endosomal acidification in eukaryotes, could also play a plasma membrane transport
role. Contemporaneous with this discovery, V-ATPases
were shown to reside on the plasma membrane of intercalated cells of the vertebrate kidney collecting duct (35).
The plasma membrane role for V-ATPase was thus not an
insect “special case” but was phylogenetically widespread: insects diverged from mammals ⬃400 million
years ago. Since then, at least 200 plasma membrane
manifestations of V-ATPase have been documented (99,
245) and are invariably associated with arrays of portasomes at the transporting membrane.
Given the two very different roles played by VATPases, it is thus of great interest to establish whether
the plasma membrane and endomembrane V-ATPases are
the same or different. The classical route to characterizing a transport process is through physiology, pharmacology, and membrane biochemistry. However, reverse genetics can be a potent adjunct to such an approach. Here,
such an approach is described in Drosophila, and the
insights it gives into V-ATPase function are outlined.
A. Drosophila vha55 Is Single Copy and Located
at 87C
The first Drosophila V-ATPase subunit to be cloned
was vha55, encoding the 55-kDa cytoplasmic B subunit
(54). Most V-ATPase subunits show very tight sequence
conservation at the amino acid level and so can readily be
cloned by homology PCR with degenerate primers (54).
Vha55 was shown to exist in single copy, but to have
Physiol Rev • VOL
multiple transcripts. It thus seemed an ideal object of
study for a genetic dissection of plasma membrane and
endomembrane roles.
B. The 87C Region Has Been Subjected to Intense
Genetic Analysis
Once a gene has been identified in a model organism,
it is important to map it cytogenetically. In this way, it
may be possible to reconcile a novel gene with a known
genetic locus previously identified by forward genetics. If
this is the case, a phenotype may already have been
described for the locus. This applies a fortiori in Drosophila, as ever since the pioneering days of Morgan and
Bridges, mutant loci have been mapped by recombination, and stocks kept alive at stock centers, in case they
proved useful in the future. (As many as half of the
mutants identified by Morgan and Bridges in the pioneering years of the early 20th century are still available in
stock centers today!) Although the presence of a sequenced genome now makes chromosomal in situs redundant, the principle of drawing inferences by bridging sequence, recombination, and cytogenetic maps remains
valid.
For vha55, the single copy gene mapped to 87C on
chromosome 3R. This has been an area of intense study,
because two copies of the heat shock gene hsp70 are
located within 87C, and the 210-kb region had been subjected to saturation mutagenesis (85) in a vain effort to
produce flies deficient in hsp70 (unknown to the experimenters at the time, there were two further copies of
hsp70 at 87A). It proved possible to show that a P-element
insertion that had been mapped cytogenetically to 87C
was in fact a disruptant of vha55. This constituted the
first documented knock-out of a V-ATPase in an animal
(54), and as the P-element insertion was lethal when
homozygous, it demonstrated the essential role of VATPases in animals.
C. P-element LacZ Reporter Reveals V-ATPase
Expression Patterns
All modern P-elements contain a reporter gene (usually lacZ) behind a weak (permissive) promoter, so conferring properties of an enhancer trap element. The elements can thus report on levels of gene expression in a
tissue. Importantly, they give information additional to
either Northern blots or in situ hybridization. Like nuclear
run-on assays, they can indicate the rate of gene transcription rather than the standing levels of mRNA. This is
because the level of an mRNA reflects a balance between
its transcription and degradation, and although it is traditional to assume that only the former process is dynamically controlled, there is no evidence that this can be
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assumed a priori. However, an enhancer trap reporter is a
heterologous gene, so its mRNA will not be processed like
that of the gene in which it inserts. There is an additional
advantage over nuclear run-on assays, because enhancer
detectors report on single cells.
Although the LacZ reporter of the lethal vha55 insertion is in opposite sense to vha55, it reports a plausible
expression pattern (Fig. 11) that has since been validated
with insertions in genes encoding other subunits (61, 65).
Vha55 is transcribed at high levels in those tissues in
which V-ATPase is thought to play a plasma membrane
role, namely, the Malpighian tubules, rectum, and the
trichoid sensilla of the cuticle. V-ATPase is also expressed
at high levels in other tissues: vha55 is expressed at
particularly high levels in the spermatheca and uterus of
the female genital tract (54). This may indicate an important role for pH in activation of the fertilized egg as it
passes along the uterus (61), a role reminiscent of the
activation of sperm as they pass along the epididymis in
vertebrates (32).
D. Correlating Functional and Genetic Maps:
V-ATPase Expression Levels
At higher resolution, it is possible to map high-level
V-ATPase expression within Malpighian tubules, and so
test the functional applicability of the genetically derived
tubule map described earlier. Within the tubule, only the
703
main segment and lower tubule stain; the initial and transitional segments do not appear to express high levels of
V-ATPase (54). This is consistent with our understanding
of fluid secretion. As discussed above, only the main and
lower tubule segments transport fluid, though in opposite
directions (171). The initial and transitional segments are
active in calcium transport (72, 73), although the vha55
enhancer detector implies that this transport does not
require high levels of V-ATPase activity.
E. V-ATPases Are Often Found in Specialized
Cell Types
Within the main segment of the tubule, only the
larger principal cells express V-ATPase at high level (54);
the intercalated stellate cells do not (Fig. 11F). This is of
profound importance for our understanding of the tubule,
because all previous work had assumed a uniform transporting cell type. It is now possible to assign electrogenic
proton transport to the principal cell and to consider the
possibility that the stellate cell does something different.
This finding has wide scope, because all Diptera (flies)
have stellate cells. Other orders of insect do not have
obvious stellate cells, but the possibility that morphologically indistinguishable cells may be functionally diverse
(as our enhancer trapping has shown for Drosophila; Fig.
6) has not yet been investigated in detail.
There is a striking parallel between the distribution
FIG. 11. Expression of V-ATPase B-subunit vha55 gene,
as reported by enhancer detector within mutagenic P-element. A: rectal pads. B: tormogen cells of trichoid sensilla of
antennal and labial palps. C: spermathecae and uterus of
female genital tract. D: Malpighian tubule. E: upper region of
anterior tubule, showing that expression is confined to the
main segment. F: within the main segment, only the larger,
principal cell nuclei stain (this LacZ is nuclear targeted).
[From Davies et al. (54).]
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
of plasma membrane V-ATPase in Drosophila tubules and
epithelia of other animals: in each case, the powerfully
electrogenic V-ATPase is confined to a particular cell
subtype within the tissue (34). In lepidopteran midgut, it
is confined to the apical membrane of specialized goblet
cells (67, 127). In insect sensilla, it is confined to the
tormogen cells of the trichoid (“hairlike”) sensilla (128).
In kidney collecting duct, it is found at high levels in
intercalated cells (4). In epididymis, it is confined to specialized cells that resemble kidney intercalated cells (32).
In fish gill, it is localized on the apical surface of pavement
cells, while in elasmobranch gill, it is found in mitochondria-rich cells (89, 218, 248). These latter results provide a
wide phylogenetic spread, confirming the generality of the
observation within the fish. In C. elegans, expression of
the 16-kDa proteolipid is confined to the rectum, the
excretory H cells, and a pair of posterior cells (174). There
thus seems to be abundant evidence that plasma membrane manifestations of V-ATPase are confined to specialized cells within an epithelium (34). Why might this be the
case? The highly electrogenic nature of the V-ATPase
might make it an uncomfortable neighbor for other transport proteins. The electromotive force for the V-ATPase
may be as high as 240 mV (60), and an electrical field of
this magnitude might compromise secondary transport
processes. The only exception to this rule is where a
secondary transport can be energized directly by the proton-motive force generated by the V-ATPase, as will be
outlined below; in this case, it is again desirable to have
the V-ATPase in a specialized cell to maximize the efficiency of the coupling. This necessity probably explains
the unique morphology of the lepidopteran goblet cells (5,
12, 59, 60).
F. The SzA Locus Provides Multiple Alleles
of vha55
In a model organism, the ability to reconcile reverse
and forward genetic information provides a potential information resource. It may prove that mutations in a
particular gene had been characterized by forward genetics, without foreknowledge of the identity of the gene. For
vha55, this proved to be the case. Two P-element insertions at 87C were obtained from stock centers, and one
was shown to be a disruptant of the vha55 gene (54). This
was then shown by complementation (Fig. 12) to be allelic to SzA, a locus previously documented in the region
(85). The earlier work provided an exquisitely detailed
description of the vha55 phenotype and constituted the
first animal “knock-out” of a V-ATPase subunit; it had the
additional advantage that the original experimenters had
been “blind” to the identity of the gene whose mutant
phenotype was being described (85).
The vha55/SzA locus had been subjected to a satuPhysiol Rev • VOL
FIG. 12. Complementation mapping of the vha55 locus within 87C.
Previous work at 87C had produced a number of lethal EMS alleles that
had been shown to map to just four lethal complementation groups,
SzA-SzD. Lethality of alleles in each group could be uncovered by
crossing to a particular combination of X-ray-induced deficiencies,
which deleted different regions of 87C. The lethal P-element insertion
was highly likely to correspond to one of these complementation groups,
so by crossing it to each of the extant deficiencies, it was possible to
work out which group corresponded to vha55. In this way, it was shown
that vha55 was allelic to SzA (54).
ration mutagenic screen, resulting in the identification of
13 ethyl methyl sulfonate (EMS) alleles (a chemical mutagen that typically induces point mutations or very small
deletions), together with two deficiencies (deletions) removing the entire gene. More than half of these mutant
alleles had been retained and were available for experimentation 10 years later. Although most alleles were recessive embryonic or larval lethal (implying that zygotic
V-ATPase function was essential from late embryogenesis
onward), some trans-heterozygotes among the alleles
were viable, giving a mild (crumpled wings) or severe
(crumpled wings, pigmentation, sterility) phenotype. Additionally, two trans-heterozygotes gave a temperaturesensitive phenotype (85).
Two aspects of the phenotype were particularly instructive. Flies homozygous for the large 210-kb deletion
Df(3R)kar3J that spanned the whole of 87C, including
vha55, died early in the first larval instar. However, the
homozygous EMS alleles showed a range of phenotypes
from embryonic lethal to subvital. How could some point
mutations be more severe than deletions of the entire
gene? The answer came later from our understanding of
the V-ATPase structure; there are three copies of the B
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subunit in every holoenzyme, and as the V-ATPase is
thought to act by a rotational model (161), all must be
essential for continued operation of the pump. There is
presumably a significant maternal investment of VATPase in the embryo, which accounts for survival to the
early first instar larva. However, if a correctly folded but
inactive V-ATPase B subunit were made, it could act as a
dominant negative, so producing a more severe phenotype than absence of the gene (54, 61, 65). We predict that
this antimorphic phenotype should be general for VATPase subunits present in multiple copies in the holoenzyme (for example, A, B, and C), not just in Drosophila,
but in any organism.
A second aspect of the Df(3R)kar3J phenotype is also
well explained in hindsight. Dying larvae have transparent
Malpighian tubules; this is autonomous in transplants of
affected tubules to abdomens of healthy flies (85). The
lumens of normal tubules usually contain crystals of uric
acid, making them look opaque white; like birds, insects
employ a uricotelic excretory system for metabolic nitrogen, to conserve water (this is discussed further in sect.
XI). The transported species is probably soluble urate, and
the Drosophila genome contains two putative urate transporters. To precipitate uric acid within the tubule lumen,
it is necessary to bring down the pH, so the apical VATPase could be expected to play a crucial role. Mutations in vha55 could thus be expected to be defective in
urinary acidification, and thus to have transparent tubules.
1. An epithelial phenotype in larvae homozygous
for lethal mutations in V-ATPase
If knock-outs of vha55 produced a transparent tubule phenotype, it seemed likely that this phenotype
could be general to mutations in all V-ATPase subunits
705
that played a role in the plasma membrane V-ATPase. A
mutation in vha68 –1, one of three tandem genes encoding the V-ATPase A subunit, was also shown to display
this phenotype (Fig. 13).
This finding has a more general significance for physiologists, as it identifies a screen, not only for other VATPase genes that play plasma membrane roles, but for
the associated chaperone, targeting and cytoskeletal proteins essential for the plasma membrane state (63). Although there is an elegant pH-sensitive lethal screen for
V-ATPase subunits in yeast (160) (that has indeed given us
most prototype subunit genes), yeast are devoid of epithelia. To be able to screen for proteins associated with
the plasma membrane role of a V-ATPase requires a multicellular model organism, and at present, the screen outlined here is the only one available.
G. A Human Renal and Auditory Phenotype Is
Associated With a Plasma-Membrane V-ATPase
The genetic analysis of V-ATPases in Drosophila has
produced results at two levels. In the insect sphere, it has
shown the role of V-ATPase in development and renal
function, outlined those tissues where V-ATPases are expressed at high levels, and those where they may play a
previously unsuspected role. At a more general level, the
first knock-out of a V-ATPase subunit has shown that
V-ATPases are essential in animals and has predicted a
crucial role in certain tissues, notably renal, reproductive,
and sensory. These predictions have since been borne out
by discovery of human mutations in V-ATPase subunits
that have renal and auditory phenotypes (121). Although
not yet documented in humans, the Drosophila results
predict that some mutations may well exhibit dominant
negative phenotypes. Although much work has proven
FIG.
13. The transparent tubule phenotype is
shared by V-ATPase mutations. Left: a dying first instar
larva, homozygous for a lethal P-element insertion in
vha68 –1. Right: its heterozygous sibling. Uric acid crystals appear dark by transmitted light. [From Dow et al.
(65).]
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
possible in human [for example, mutations of V-ATPase
genes expressed predominantly in kidney show a distal
tubular acidosis phenotype but not sensorineural deafness (211), so distinguishing V-ATPase roles], human genetics remains serendipitous. It is thus likely that deeper
understanding of the plasma membrane V-ATPase complex can more easily be achieved in a model organism.
IX. HOW DO CHLORIDE AND WATER CROSS
THE TUBULES?
The insect Malpighian tubule secretes fluid faster
than any other epithelium on a per-cell basis (63, 66).
Accordingly, one might expect highly specialized adaptations for shunt conductance and water flux. Classically, it
has been considered that chloride moved passively
through a paracellular route in Dipteran tubule (239). In
this section, we develop the argument that, at least in
Drosophila, chloride and water conductance are both
transcellular through just the stellate cells of the main
segment of the tubule. In this way, the active transport
sites of high metabolic activity (the principal cells) can be
separated from the passive sites of high flux (the stellate
cells). This spatial segregation may represent a specialization for very high transepithelial flux rates, or it may be
a consequence of the driving force being provided by
V-ATPase, a pump invariably consigned to specialized
subsets of cells (see sect. XE).
A. Leucokinins Selectively Increase
Chloride Conductance
In most insects studied, it appears that tubule chloride shunt conductance is controlled by the tachykininlike peptide leucokinin (102), discussed in more detail in
section XI. This peptide provides an excellent tool for the
selective manipulation of the chloride shunt. In Drosophila there is a single leucokinin (Asn-Ser-Val-Val- Leu-GlyLys-Lys-Gln-Arg-Phe-His-Ser-Trp-Gly-amide), encoded by
the pp gene (226). Leucokinin has three separately measurable effects: 1) it acts rapidly on intracellular calcium
in just stellate cells (226), with a maximal response within
100 ms and a EC50 between 10⫺11 and 10⫺10 M. 2) Fluid
secretion also rises rapidly (as fast as can be measured)
with a similar EC50. 3) As in other insects, the target of
leucokinin action is thought to be the chloride shunt
conductance, as the tubule’s lumen-positive potential is
collapsed toward zero on application of leucokinin. This
is a rapid event, complete within 1.5 s (170). 4) Consistent
with these results, the Drosophila leucokinin receptor has
recently been characterized and has been shown to have
an EC50 between 10⫺11 and 10⫺10 M, and to act through
intracellular calcium, in cultured cells. Additionally, the
Physiol Rev • VOL
receptor is only found in stellate cells within the tubule
(181).
B. Patch Clamp Reveals Maxi-Chloride Channels
in Tubules
If chloride moves transcellularly, then it would be
expected that chloride channels with defined pharmacology could be detected. This is the case: fluid secretion is
sensitive to a range of classical chloride channel blockers,
at concentrations similar to that effective in vertebrate
systems (172). In addition, maxi-Cl channels (Fig. 14),
with similar properties to those observed in a range of
human tissues (36, 110, 156), are found in apical membrane patches (172). These channels are DIDS sensitive,
with a large unit conductance of 280 pS (172). Consistent
with the action of leucokinin to raise calcium, vertebrate
maxi-Cl channels are known to be Ca2⫹ sensitive (36, 110,
156).
Maxi-Cl channels could only be found in a small
fraction (2/128) of electrically good patches, although
they were abundant in each patch where they were found,
suggesting that only a small fraction of the apical membrane contained such patches. Naturally, this suggested
the stellate cell apical membrane as a candidate.
C. Self-Referencing Electrode Analysis Shows That
Chloride Moves Through Stellate Cells
Self-referencing electrode analysis revealed a relatively small number of hotspots along the tubule (Fig.
15A), and these all mapped to the main segment (see sect.
IXA4), the site of fluid secretion (see sect. VIIA8). When
hotspots were mapped at high resolution, they were invariably found to be centered on stellate cells (Fig. 15B).
FIG. 14. Maxi-chloride channels in apical patches of Drosophila
Malpighian tubule. [From O’Donnell et al. (172).]
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FIG. 15. Chloride flows through stellate cells. A: vibrating probe survey of an
anterior tubule, initial segment to the left
and ureter to the right. Current hotspots
are confined to the main segment of the
tubule. B: higher power view of a current
hotspot, showing the currents are centered over a stellate cell. C: chloride substitution experiment, showing that current is carried by chloride. D: niflumic
acid, a chloride channel blocker, abolishes chloride current hotspots. Numbers show the current at different times
(in min) from the application of niflumic
acid. [From O’Donnell et al. (172).]
In principle, these currents could be carried either by
an influx of chloride or an efflux of a cation. However, ion
substitution experiments confirmed that an influx of chloride was responsible (Fig. 15C), and this was confirmed
by abolishing hotspot current with chloride channel
blockers (Fig. 15D). It thus seems clear that, in Drosophila, chloride flows through channels of classical pharmacology, located in the stellate cells of the main segment.
There is support for this transcellular flux model in
other species. Recently, chloride channels were shown in
Aedes (mosquito) Malpighian tubule, although these were
lower conductance than in Drosophila (169). This may
reflect a genuine difference in that chloride was considered to flow mainly paracellularly in Aedes tubules (178,
239). In Drosophila, additional routes for chloride flux
have been proposed. A bumetanide-sensitive K⫹/Cl⫺ cotransport was demonstrated in basolateral membrane of
principal cells (142); as this is electroneutral, its presence
would not be detected by vibrating probe. A sodiumdependent Cl⫺/HCO⫺
exchanger, NDAE1, has been
3
cloned and shown to be expressed basolaterally in Malpighian tubule (206). The relative contribution of this
exchanger to tubule secretion has not yet been determined. The relative significance of these routes for chloride flux has not been established; however, Cl⫺ channel
blockers abolish transport (172), whereas bumetanide
only attenuates it (142), implying that, in Drosophila, the
stellate route predominates.
D. Tubules Contain CLC Chloride Channels
The molecular identity of the chloride channels in
tubules is not yet known. However, an advantage of working in a model organism is that candidate genes can be
reliably identified in silico. The Drosophila genome conPhysiol Rev • VOL
tains three chloride channels (CG6942, CG8594, and
CG5284) of the CLC family (Fig. 16). They are evenly
spread among the major branches of the human CLC
family, suggesting that single genes cover the major roles
of the family. There is a clear pattern that the Drosophila
genome will frequently have a single gene, perhaps with
multiple transcripts, corresponding to typically three similar human genes.
If one of these genes were expressed uniquely in
tubules, that would be strong evidence for a candidate
locus for an epithelial chloride channel. However (Fig.
16), all three are expressed in tubule. So further analysis
will be needed to establish which may play epithelial
roles.
E. Bartter Syndrome Type III
Although there are not any extant mutants of the
three CLC genes, human studies of CLC mutations may in
this case be informative for Drosophila. Bartter syndrome
is an autosomal recessive form of (often severe) intravascular volume depletion, due to renal salt-wasting. Patients
with Bartter syndrome are often critically ill from birth
onward, and their long-term clinical course is also frequently complicated by nephrocalcinosis, leading to renal
failure (115, 188). The syndrome encompasses three major classes of mutation in renal transporters or channels;
Bartter syndrome type III corresponds to mutations in the
CLCNKB gene, encoding a kidney-specific CLC, that impair renal chloride reabsorption in the thick ascending
limb of Henle’s loop (209). Given the relative facility with
which genes can be mutated in Drosophila, it will be
interesting to establish whether similar symptoms are
associated with mutations in any of the Drosophila CLCs.
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
FIG. 16. Tubules express all Drosophila CLC channels. A: similarity tree
for the human CLC family and all Drosophila members identified by TBLASTN
search against the completed Drosophila
genome, using human CLC1 as probe.
There are single Drosophila representatives in each major group of the CLC
family. B–D: all Drosophila members of
the CLC family are expressed in tubule.
RT-PCR was performed using intronspanning primers, using the following
templates: genomic DNA (lane 2), head
cDNA (lane 3), tubule cDNA (lane 4), no
template control (lane 5). DNA ladder is
in lane 1 of each figure. The genes detected are as follows: B, CG5284; C,
CG6942; D, CG8594. In each case, a
product of the expected (cDNA) size is
seen in the tubule lane. (From P. Stockinger and J. A. T. Dow, unpublished
data.)
F. How Does Water Cross the Tubule?
An interesting “back-of-the-envelope” calculation: the
volume of cytoplasm in the active region of the tubule is
⬃1 nl, and the maximum secretion rate we have observed
is 6 nl/min. This means that the fully stimulated tubule
pumps its own volume every 10 s! This is very similar to
the result obtained for the Rhodnius tubule (153), implying this rate is a characteristic of insect tubules. Such
extremely high flux rates pose potential problems. Could
cells withstand such high transit rates in the steady state?
Could they withstand volume transients as transport was
up- or downregulated? There is clearly a benefit in protecting the actively transporting principal cells from rapid
fluxes. This could be achieved either by paracellular flux
or by aquaporin-mediated flux through a subset of cells in
the epithelium. There is no rigorous information yet, but
Physiol Rev • VOL
fluid secretion by tubules is known to be sensitive to
mercuric ions and organo-mercurials, the best available
inhibitors of aquaporins. In silico, it is possible to identify
a large gene family of Drosophila aquaporins, and most of
these are expressed in tubules. Consistent with this, stellate basolateral membrane is stained prominently by antibodies to human aquaporin-3 (Fig. 17) or to buffalo fly
aquaporin. If this localization were confirmed, it would
provide an elegant solution to the problems of withstanding high flux rates through the tubule.
G. Cation and Anion Transport Are
Spatially Separated
It is thus clear that active, electrogenic cation transport is spatially segregated from passive chloride shunt
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709
FIG.
17. Antibodies to vertebrate
AQP3 selectively label stellate cells. Four
typical views are shown. (From D.
Brown and J. A. T. Dow, unpublished
data.)
conductance, and probably from water flux, in this epithelium. This may confer particular advantages in the
spatial optimization of a tissue subjected to uniquely high
flux rates, as the cell with intense transport activity, and
commensurate intense metabolism, can be shielded from
a massive transcellular flux that might compromise its
function. Chloride, and we hypothesize also water, could
then flow through a far less metabolically active cell,
which has the less onerous task of merely staying alive.
This is reminiscent of other systems in which the VATPase is hypothesized to play a plasma-membrane role
and is found in specialized cells (34).
X. PHARMACOLOGY AND CELL
SIGNALING MECHANISMS
A. Cell-Specific Calcium Signaling
Calcium signaling and transport processes regulate cellular processes and contribute significantly to renal function
(82); furthermore, kidney disease is often associated with
channel abnormalities that affect epithelial transport (203).
However, there is not a thorough understanding of the complex contribution of calcium homeostasis to vertebrate renal
function due to the lack of genetic models for fluid-transporting epithelia. The importance of the Drosophila tubule
in such studies is clear: 25–30% of the human proteome
constitutes signaling and transport proteins (110a); most of
these will have Drosophila tubule homologs. Furthermore,
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at least 11 Drosophila genes show significant similarity to
human renal disease genes (194), suggesting the utility of the
tubule in studies of signaling and renal disease.
The importance of calcium signaling and transporting mechanisms in the tubule is demonstrated by the
storage of calcium concretions in tubules in many species, including Drosophila (243), Rhodnius (149), and
Calliphora (222). Furthermore, Malpighian tubules contain the highest amounts of calmodulin compared with
other tissues (249). This suggests that tubules are the main
site of calcium storage, and thus may also be the tissue with
the highest turnover of calcium (223). More recent work in
Drosophila has shown that anterior tubules contain 25–30%
of the calcium content of the whole fly (73). Thus, apart from
its role in osmoregulation, the tubule is critical to the maintenance of whole body calcium homeostasis.
To define the contribution of calcium signaling processes to renal function, it is necessary to obtain direct
measurements of intracellular cytosolic Ca2⫹ levels
([Ca2⫹]i). Calcium-binding fluorescent dyes are extruded
from insect tubules very rapidly, thus preventing measurements of [Ca2⫹]i (J. A. T. Dow and T. Cheek, unpublished observations). The extrusion of fluorescent molecules by tubules is thought to be due to the activity of
P-glycoprotein, or a related transporter (29). Furthermore, tubule cells are too small to allow reliable measurements by ion-selective microelectrode impalements to be
made. Accordingly, a targeted Ca2⫹ reporter was developed in Drosophila to allow organotypic [Ca2⫹]i measurements in intact, viable tissue. The luminescent jellyfish
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
Aequorea victoria utilizes a photoprotein, aequorin, and
produces flashes of blue light that are transduced to green
by green fluorescent protein. Critically, the intracellular
signal used to elicit this flash in vivo is an increase in
[Ca2⫹]i. Because the dynamic range of [Ca2⫹]i concentrations in jellyfish is very similar to those in plants and
animals (generally 50 –500 nM), this means that aequorin
is a useful calcium indicator.
Transgenic aequorin was first used in plants (130), but
the UASG-GAL4 system for targeted expression (see sect.
IXA7) allowed a generic indicator to be constructed that
could be driven by any GAL4 line of interest. Transgenic
Drosophila that express (apo)aequorin under UASG control
were generated; these lines were crossed into lines expressing appropriate tubule-specific GAL4 drivers (214). This results in expression of the aequorin transgene in specified
tubule cell types or tubule regions (190) (Fig. 18), which
allows analysis of calcium cycling mechanisms in vivo.
Monitoring of [Ca2⫹]i increases induced by the ER
Ca2⫹-ATPase inhibitor thapsigargin, in either principal or
stellate cells (Fig. 19), showed that only stellate cells
maintained this response in calcium-free medium. This
result indicates that in principal cells, the thapsigargininduced [Ca2⫹]i rise is dependent on extracellular calcium
and that calcium stores are emptied too rapidly to be
monitored, or that these are too small. In stellate cells,
however, significant calcium extrusion from ER stores occurs, which suggests a role for “capacitative calcium entry”
in this cell type. The novel and important observation in
Drosophila that adjacent cells in transporting epithelia may
operate distinct calcium cycling mechanisms may well extend to vertebrates; however, this can only occur when
technological limitations have been overcome.
B. Neuropeptide-Stimulated Calcium Signaling
Successful targeted expression of aequorin in Drosophila allowed the first organotypic measurements of
FIG. 18. Cell-specific real-time calcium measurement using UAS-directed apoaequorin in Drosophila. A–E: different
tubule regions identified by the GAL4 driver lines in the left column. F: overview of the Malpighian tubule. G–J:
corresponding patterns of expression for each GAL4 driver, visualized here with UAS-GFP. K–P: real-time calcium
recordings. These provided the first basal calcium measurements in an insect tubule and showed that the neuropeptide
CAP2b acted only on principal cells in only the main segment of the tubule, whereas leucokinin acted only on stellate
cells (inset in M). [From Rosay et al. (190).]
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711
FIG. 19. Thapsigargin unveils distinct calcium pool kinetics in adjacent cell types. The
effects of thapsigargin on fluid secretion (A
and D) and on the calcium levels in principal
(B and E) and stellate (C and F) cells are
shown in the presence (A–C) and absence
(D–F) of external calcium. Although thapsigargin stimulates fluid production irrespective
of the presence of calcium, the type I cells
only show a calcium response when external
calcium is present. [From Rosay et al. (190).]
tubule [Ca2⫹]i in any insect. These experiments demonstrated a novel role for the nitridergic neuropeptide
CAP2b [pyro-ELYAFPRVamide (53) and below] in renal
calcium signaling (190) and showed that CAP2b stimulated an increase in [Ca2⫹]i in a dose-dependent manner,
which only occurred in principal cells in the main, fluidsecreting segment. Elevations in [Ca2⫹]i were correlated
to CAP2b-induced increases in fluid transport rates. FurPhysiol Rev • VOL
thermore, both CAP2b-induced [Ca2⫹]i and secretion are
dependent on extracellular calcium (Fig. 20).
The diuretic neuropeptide leucokinin induces fluid
secretion via activation of calcium signaling mechanisms
in several insect species (63). However, direct demonstration of the activation of calcium signaling was achieved
using targeted aequorin in Drosophila tubules (190). Leucokinin stimulates a rapid millisecond rise in stellate cell
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
FIG. 20. CAP2b signaling relies on external calcium. A–D:
principal cell calcium signal elicited by CAP2b stimulation is
dependent on external calcium. As bathing calcium levels are
reduced, the signal becomes smaller, until it is abolished in
nominally Ca2⫹-free saline. D and E: stimulation of fluid secretion by CAP2b requires external calcium. Tubules were stimulated with CAP2b at 70 min, in the presence (gray squares) or
absence (black diamonds) of external calcium. [From Rosay et
al. (190).]
[Ca2⫹]i from a resting concentration of 80 –200 nM (172).
Leucokinin also activates chloride shunt conductance in
stellate cells, and chloride channel blockers inhibit leucokinin-stimulated fluid secretion (172). More recently, the
gene encoding Drosophila leucokinin, pp, was identified,
and the Drosophila peptide (Drosokinin) isolated (226).
Expression of pp has been detected in Drosophila heads
(226), suggesting that Drosokinin is synthesized in the
central nervous system and released into the hemolymph.
Drosokinin elevates [Ca2⫹]i in only stellate cells at an
EC50 between 10⫺10 and 10⫺11 M to induce high rates of
fluid secretion.
C. Calcium Channels
Both anterior and posterior tubules in Drosophila
perform transepithelial calcium transport (72). However,
the demonstration of a large basolateral calcium flux
(entry of calcium into cells via the basolateral membrane)
compared with a smaller transepithelial flux (transport of
calcium across the epithelium into the lumen) suggests
that most of the Ca2⫹ entering the cell is sequestered
within it. Thus plasma membrane calcium entry channels
must play an important role in maintaining both calcium
and fluid transport by the tubule. Recently, L-type calcium
Physiol Rev • VOL
channels, most often associated with neuronal cells (9) in
both insects and vertebrates, have been shown to modulate epithelial fluid transport. Similarly, L-type calcium
channels are found in vertebrate kidney cells and are
associated with calcium reabsorption (256), transepithelial calcium flux (83), and mechanical stress (257); thus
L-type channels are not confined to excitable cells.
There are three L-type ␣-subunit genes in the Drosophila genome: Dmca1A (night-blind), Dmca1D, and a
novel gene, CG1517. The classical L-type channel blockers, verapamil, and the dihydropyridine nifedipine inhibit
CAP2b-stimulated fluid transport and calcium influx in
principal cells (146). The inhibition of response by vertebrate channel blockers suggests conservation of channel
function during evolution. Immunolocalization of ␣-subunits indicates expression only in main segment, both at
the basolateral and apical surface. Use of fluorescent
conjugates of verapamil and nifedipine in unfixed, intact
tubules reveals both high- and low-affinity sites for verapamil binding in main segment, with low-affinity sites
present in basolateral membranes and high-affinity sites
located in apical microvilli. However, a major site of
dihydropyridine binding occurs in the initial segment, a
major site of calcium transport and storage (72, 73). Thus
L-type calcium channels not only facilitate neuropeptide-
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mediated transport and calcium signaling in principal
cells, but it is likely that these also have a role in transepithelial calcium transport.
Most recently, a novel epithelial role for the transient
receptor potential (TRP) family of calcium channels (94)
has been described. TRP represents a highly calciumselective cation channel, whilst trpl (trp-like) encodes a
nonselective cation channel with moderate calcium permeability. The TRP channel family was first identified in
Drosophila, and ⬃20 mammalian TRP proteins have since
been identified, which fall into at least three subfamilies.
Much of our current understanding of the functional role
of TRP in vivo is derived from Drosophila photoreceptors
(93), where the light-sensitive current in photoreceptors
is completely abolished in trpl:trp double mutants lacking
both TRP and TRPL (162, 185). Response to light is dependent on the close interaction of TRP and TRP-related
channels with other signaling proteins (rhodopsin, phospholipase C, protein kinase C, calmodulin) mediated
through the scaffolding protein INAD (inactivation noafterpotential D) (93). Recent work has shown that INAD
is required for correct localization of TRP-containing supramolecular complexes in the eye (140). Additionally, a
newly identified third member of the TRP family, TRP-␥,
may form heteromultimers with TRPL (251). While much
of the work on vertebrate TRP channels has been predominantly based in cultured cell models, the work on
Drosophila TRP/TRPL has been performed almost exclusively in an organotypic context.
The role of TRP and TRP-like channels in nonvisual
systems is poorly understood. Recently however, a role
has begun to be elucidated in transporting epithelia (147).
Molecular data show that genes encoding TRP, TRPL, and
TRP-␥ are expressed in Drosophila tubules. TRP has been
immunolocalized to main segment, while TRPL is located
throughout the tubule, suggesting roles for TRPL in both
calcium transport and signaling. Using mutant alleles of
trp and trpl, we have shown that unlike phototransduction, which is dependent on TRP, neuropeptide-stimulated fluid transport is critically dependent on TRPL. Significantly, tubules do not express INAD, suggesting that
while transporting epithelia may utilize the same proteins
as excitable cells, the complexes formed, if any, are distinct. Also, as INAD has been shown to be critical for TRP
function in the eye, the work in tubules suggests that this
must be a tissue-specific, rather than a general, requirement for TRP function.
Thus, while the study of calcium signaling events in
an organotypic context may be intrinsically difficult, the
insights generated are particularly valuable and only obtainable in this way. It is clear that renal epithelia utilize
a range of calcium signaling mechanisms that are at least
as complex as those in the neuronal context and that
these operate in concert with the reabsorption and transepithelial transport of bulk calcium.
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713
D. NO/cGMP Signaling
The mobilization of NO/cGMP signaling is a potent
mechanism for the control of cell and tissue function. The
NO signaling pathway is implicated in neuronal, immune,
vascular, and renal function in vertebrates and is so highly
conserved throughout evolution that these physiological
processes are also NO regulated in insects (52). However,
very little is known about NO-mobilizing peptide hormones, especially in relation to renal function. However,
the role of cGMP in kidney function, including renin
secretion, is beginning to be understood, especially in
relation to guanylin action (81). Furthermore, the cellular
distribution of NO synthase (NOS) isoforms (13) and
soluble guanylate cyclase, cellular targets for NOS-generated NO, have been mapped in kidney (228), although
direct demonstrations of NO/cGMP modulation of renal
function have not been performed in vertebrates.
In insects, application of exogenous cGMP has been
shown to modulate diuresis, in either a stimulatory (Drosophila) (57) or inhibitory (Rhodnius) (180) capacity. The
stimulatory role of NO-generated cGMP has also been
documented in tubules, via use of NO donors (57).
The only known example of nitridergic peptides in
insects to date remains the capa family of peptides. We
have identified the gene capability (capa) (123), which
encodes three capa peptides, two of which (capa-1, GANMGLYAFPRV-amide; and capa-2, ASGLVAFPRV-amide)
are structurally and functionally similar to CAP2b. Both
capa-1 and capa-2, like CAP2b, stimulate fluid secretion
via [Ca2⫹]i elevation in principal cells. All three peptides
activate Drosophila NOS (DNOS), which results in elevation of cGMP levels. Moreover, capa-induced fluid transport is significantly reduced by methylene blue, an inhibitor of soluble guanylate cyclase, supporting the previous
observation that NO-generated cGMP plays a significant
role in diuresis (53). Four pairs of capa-producing neuroendocrine cells have been identified, one pair in the
subesophageal ganglion and the other three in the abdominal neuromeres; this suggests that the capa peptides are
released into the hemolymph and act as endogenous neuropeptide modulators of NO signaling (123).
The NO/cGMP signaling pathway has been extensively investigated in tubules. Tubules express DNOS,
encoded by a single gene that shares extensive identity
with vertebrate NOS1 (52). Intriguingly, NOS is only expressed in principal cells (52). At physiological concentrations, capa-induced increases in intracellular calcium
and cGMP signaling are confined to principal cells (123);
this, together with the principal cell localization of NOS,
suggests that capa-activated NO is an autocrine signal in
this cell type.
Investigation of guanylate cyclase (GC) expression in
tubule shows that several transcripts, encoding soluble
(sGC) and membrane-bound receptor (rGC) forms of the
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
enzyme are expressed. These are Gycalpha99B and
Gycbeta100B, encoding ␣- and ␤-subunits of sGC, and a
novel rGC gene, CG1073 (V. P. Pollock, J. A. T. Dow, and
S. A. Davies, unpublished data). Thus tubules contain
bona fide intracellular targets for NO action.
Downstream targets for cGMP include cGMP-dependent protein kinases (cGKs) and cGMP-dependent phosphodiesterases (cG-PDE). Tubules express two genes for
cGK, dg1 and dg2 (57). Interestingly, mutations in dg2 result
in a transport phenotype which displays hypersensitivity to
cGMP (Fig. 21) but not to leucokinin signaling, which is
confined to stellate cells. Thus cGK-induced modulation of
fluid transport is a result of signaling events in the principal
cells in the main segment. In this cell type, the ultimate
target of cGMP and CAP2b in tubule may be the V-ATPase
(see sect. IX), because an increase in transepithelial potential
is associated with cGMP or CAP2b treatment (53).
Inhibitors of PDE activity also affect fluid secretion
rates; Zaprinast, which inhibits cG-PDE (PDE5) in vertebrates, accelerates responses in fluid transport when applied to tubules (68). Thus downstream activation of
cG-PDE is an important control mechanism for fluid
transport rates.
E. Cross-talk Between NO/cGMP and
Calcium Signaling
2⫹
FIG. 22. Summary of the CAP2b/Ca /nitric oxide (NO)/cGMP signaling pathway in principal cells. See text for details.
Interactions between cGMP and calcium signaling
pathways have been observed in several vertebrate sys-
FIG. 21. Mutations in the Drosophila cGK, dg2, confer hypersensitivity to cGMP. Tubules were stimulated with 3 ␮M cGMP at 30 min and
1 ␮M leucokinin at 70 min. forS, a mutation in cGK gene dg2, produces
hypersensitivity to cGMP signaling, while leucokinin signaling in the
same tissue is unaffected. (From S. H. P. Maddrell, S. A. Davies, and
J. A. T. Dow, unpublished data.)
Physiol Rev • VOL
tems, including the visual system (133) and smooth muscle (1). However, although the role of cross-talk in renin
secretion has been under investigation (14), the direct
effects of cGMP/calcium signaling mechanisms in a renal
context have not been fully explored.
In tubules, however, exogenous cGMP has been
shown to induce a slow rise in [Ca2⫹]i levels in principal
cells, which is dependent on extracellular calcium (148).
This calcium signal is verapamil and nifedipine sensitive.
Interestingly, cGMP-induced fluid transport is also sensitive to these L-type calcium channel blockers, which suggests that calcium signaling mechanisms contribute to
cGMP-induced fluid transport. Expression of the Drosophila gene encoding a cyclic nucleotide gated channel,
cng, has been demonstrated in tubules, and it is possible
that cGMP-induced transport and calcium responses occur via a CNG channel (Fig. 22). Drosophila CNG is most
closely related to vertebrate CNG3, which is highly expressed in non-sensory tissue, including kidney (107).
Thus a role for CNG in transporting epithelia is conserved
across evolution.
In summary (Fig. 22), the Drosophila tubule contains
all the signaling components of the NO/cGMP pathway
and remains the only transporting epithelium in which a
direct role of NO/cGMP on ion transport has been demonstrated.
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F. New Targets for Drug Discovery
Although most pharmaceutical research is at present
devoted to analysis of gene products in simple cell-culture
models, there is a wide range of diseases where candidate
genes may rely for their normal function on other gene
products. In this case, analysis of the candidates demands
a heterologous expression system that can offer an organotypic context in which the transgene could be expressed. In tubules, there exists a panel of enhancer trap
lines that can direct expression of any gene of choice to
subsets of cells within the tubules, allowing us to study
the physiological phenotype of mutant genes in the context of an otherwise normal fly (see sect. VIIIA7), exactly
as we proposed to be required for integrative physiology
(see sect. III). In particular, the system can be used to
express human transport and cell signaling genes ectopically in Drosophila tubules, allowing their mutagenesis
and further detailed study. So far, we have successfully
expressed two classes of G protein-coupled receptor in
tubule cells and have shown that fluid secretion by such
transgenic tubules is then sensitive to the appropriate
ligands (125). This offers the opportunity both to study
the human genes in a heterologous, yet epithelial, context
and to manipulate second messenger pathways in specified cell subpopulations of an epithelial model.
715
gen from the purine pathway is excreted as insoluble uric
acid, thereby saving water. However, in practice, terrestrial insects diverge from this ideal: they can excrete the
bulk of their nitrogen as a range of compounds, including
uric acid, allantoin, urea, and ammonia (43). There is no
obvious phylogenetic or life-style trend underlying the
preferred compound. In Drosophila, the question is particularly intriguing, as it has a uric oxidase (uricase) gene,
that catalyzes the conversion of uric acid to allantoin
(237). Urate oxidase (UOX) is known to be expressed in
adult eye and in main segment principal cells of Malpighian tubules of third instar larvae and adults only (84).
The role in eye is specialized: storage excretion of uric
acid allows a white reflective lining for each ommatidium,
and UOX presumably plays a role in regulating the deposition of uric acid. The tubules are probably the major
sites of nitrogenous excretion and are known to be loaded
with uric acid crystals. So why is UOX expressed, and
why at only certain times of life? The situation is complicated further by work on related species: in Drosophila
pseudoobscura, UOX is expressed only in adult, whereas
in Drosophila virilis, it is expressed only in third instar
larva (238). To explain this apparently random assortment
of facts, it is helpful to take a comparative view of purine
metabolism in mammals.
A. Human, Mouse, and Gout
G. Insect Tubules as Targets
for Selective Insecticides
Insect tubules have long been identified as missioncritical parts of an insect’s physiology that might be good
targets for insecticides. Our work has shown that Drosophila tubules share close functional similarity with
those of all the 30 or so species of insect so far studied. In
particular, their function is effectively identical to those
of other Diptera. This means that Drosophila tubules
provide a useful model for those of major medical and
agricultural pests, such as mosquitoes and tsetse flies, in
advance of any transgenic technologies that may be developed for their detailed study later. They can thus act as
a proxy for species like Anopheles gambiae with a recently completed genome (27), but technically challenging transgenesis (10). This is discussed in section XIIIB.
XI. IN SILICO MAPPING
OF METABOLIC PATHWAYS
In an organism with a sequenced genome, it is possible to investigate problems at a theoretical level with a
precision not heretofore possible. As an example, we will
consider the question of purine metabolism. As outlined
in section IXF1, terrestrial insects are considered to have
a uricotelic excretory system, in which metabolic nitroPhysiol Rev • VOL
In mammals, there are two parallel pathways for
purine metabolism: UOX (usually concentrated in the ribosomes of the liver) and hypoxanthine guanine phosphoribosyl transferase (HPRT). In humans and other primates, UOX is a single copy gene, rendered dysfunctional
by two mutations. Accordingly, purine catabolism is
through the HPRT pathway, and disruption of this pathway leads to Lesch-Nyhan syndrome, with mental retardation, cerebral palsy, choreoathetosis, uric acid urinary
stones, and self-destructive biting of fingers and lips (136).
Partial deficiency can lead to Kelley-Seegmiller syndrome,
featuring renal stones, uric acid nephropathy, renal obstruction, and gout (124).
In contrast, in rodents, UOX is functional and the
HPRT pathway is insignificant. Accordingly, mutation of
rodent UOX leads to neurological symptoms highly reminiscent of Lesch-Nyhan syndrome (250). These findings
show that there are alternative, potentially redundant,
pathways of purine catabolism; it is only the overall function that is necessary for life.
B. Drosophila, Mice, Humans, and Gout
In the three Drosophila species, it is highly unlikely
that UOX is essential, since closely related species express it at different life stages and have a visibly func-
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
tional uric acid excretory pathway. The most likely explanation is that the UOX pathway to allantoin provides extra
capacity at life stages appropriate to the organism’s lifestyle. Insects generally feed most intensively in their last
larval instar, and scaling arguments imply that problems
in excretion are exacerbated by size. It is thus not unreasonable to assume that this semi-redundant pathway
gives a slight selective advantage that is decisive in wild
populations.
An alternative explanation, offered for both human
and Drosophila, is that urate is protective under conditions of oxidative stress. There is experimental evidence
in favor of the model in Drosophila: mutants deficient in
synthesis of urate showed reduced tolerance to oxidative
stress from any of three sources (paraquat, ionizing radiation, and hyperoxia) (104). Furthermore, synthetic lethality was obtained by crossing urate-deficient flies with
superoxide dismutase (SOD) mutants, implying that both
pathways provide partial protection against oxidative
stress (104). This is only a partial explanation, because it
still does not explain why different, closely related species of Drosophila have very different expression patterns, or why just the tubules and eye should need to be
afforded such extra protection. However, these results
show that urate provides real protection against oxidative
stress in Drosophila.
to the kidney, such as urethral lithiasis (154) and hydronephrosis (155). In Drosophila, severe rosy mutants have
shortened and malformed Malpighian tubules that contain
yellow to orange-colored globular inclusions in the lumen
(92). Like their human counterparts, ry flies are sensitive
to dietary purines (198). Untreated, severe ry alleles are
semilethal and temperature sensitive. It is thus clear that,
despite apparently gross differences between their anatomy, the cause and symptoms of xanthinuria type I are
conserved between human and fly.
However, there is an added value to fly work compared with human observational studies. In humans, the
penetrance of mutant phenotypes can vary widely, confounding systematic study. This is presumed to be because of variation at other predisposing loci. In Drosophila, it is possible to test for interactions between loci by
controlled crossing. Accordingly, rosy mutations have
been crossed to similar eye color mutants, and synthetic
lethality has been observed in crosses with cinnamon
(cinn), low xanthine dehydrogenase (lxd), and maroonlike (mal) (87). The molecular identities of these loci are
not all known, but they presumably encode cofactors of
rosy that together are essential for normal function. Such
results may thus provide novel insights into the distressing group of human xanthine metabolism disorders (7).
E. Virtual Metabolomics: an Index of Possibilities
C. Disorders of Purine Metabolism
Obviously, it would be of interest to extend the mutational data on UOX to Drosophila, but there are no
known Drosophila UOX mutants. However, purine metabolism is a multistep pathway, and it is reasonable to
assume that mutations at other key steps in the pathway
might elicit similar symptoms to a UOX blockade. In
vertebrates, urate is generated by xanthine oxidase, and
defects in this enzyme lead to xanthinuria type I (55),
characterized by low serum urate and very high levels of
urinary xanthine, frequently forming yellowish stones.
These stones can lead to obstructive nephrolithiasis, and
the best treatment is removal, combined with high water
intake and dietary modification.
D. Does Drosophila Have a Xanthine
Oxidase Mutant?
Interestingly, Drosophila does have a xanthine oxidase mutant; it is the eye-color mutation rosy (ry), first
characterized by Bridges, and the subject of 612 papers.
The phenotypes of human and fly mutations are strikingly
similar. In human patients, there are low serum and urinary levels of urate and very high levels of xanthine,
causing formation of yellowish xanthine calculi (55).
These frequently cause distortion and obstructive damage
Physiol Rev • VOL
The principle illustrated here with xanthine oxidase
and urate oxidase in Drosophila can readily be extended.
It is possible to sketch out in silico a potential metabolic
pathway for purines, or for any pathway of interest, and
interrogate the genome to see whether any candidate
gene exists for a given step in the pathway. This principle
suggests fast and easy experiments, for example, primer
design for RT-PCR, to test which elements of a pathway
are present in a tissue of interest. Although this procedure
falls far short of a proof that a particular pathway operates as outlined in that tissue, the absence of a particular
gene in the genome, or the absence of expression of a
gene at levels detectable by PCR, would be powerful
negative evidence. This technique, which we name “virtual metabolomics,” gives rapid insights into an index of
metabolic possibilities in a tissue of interest. It is, of
course, only as authoritative as the genome being analyzed, and so is presently most useful in human and other
genetic model organisms for which complete sequence is
compiled.
The application of this technique to the purine metabolism pathway in Drosophila is shown in Figure 23.
This analysis can be reconciled with physiological
data. At different stages in its life cycle, different genes
are expressed. Thus, in the absence of urate oxidase
(expressed only in third instar larvae and adults), it is not
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717
FIG. 23. Virtual metabolomics of the purine metabolism pathway in Drosophila. A: part of the Kyoto Encyclopedia
of Genes and Genomes (KEGG) display for purine metabolism (120). B: reconstruction of the possible fate of xanthine
in normal and mutant Drosophila tubules.
possible to form allantoin, so uric acid is likely to be the
only by-product. In contrast, in third instar and adult,
allantoin can be produced, and the presence of an allantoinase implies that urea could be formed.
The analysis also highlights the impact of mutations
in diverting metabolites with pathological consequences.
In rosy flies, xanthine oxidase is absent, so xanthine
accumulates to levels that produce xanthine stones. When
V-ATPase is defective, then although urate is formed, it
does not precipitate as luminal uric acid.
Physiol Rev • VOL
F. Eye Color Mutants Have Interesting Causes
The first Drosophila mutation described (white) was
for eye color (158); appropriately enough, the gene encodes an ABC transporter with both eye and Malpighian
tubule transport phenotypes, and now over a hundred
distinct loci are known that produce an eye color phenotype. Interestingly, many of the eye color mutants have
tubule color phenotypes (242); indeed, most classical
Drosophila geneticists would consider the tubule as a
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
larval indicator that predicts adult eye color. The reason
for the congruence between eye and tubule is not entirely
clear, but it may reflect an original excretory function of
the pathways that generate and transport the major
classes of metabolite (ommochromes and pteridines) that
were subsequently pressed to a specialized role in pigment granule formation. [An alternative explanation, that
purine metabolism may allow urate to accumulate and
provide protection against oxidative stress, has been outlined above (104).]
With the benefit of the genome project, it has recently
proven possible to reexamine the massive literature on
eye color mutations (there are 2,374 references for the
white gene alone) and assign them to one of three broad
classes. These are as follows: metabolic/biosynthetic enzymes that form the pigments or their precursors; transporters, frequently of the ABC class, that move pigments
or precursors to their targets; and vesicle trafficking proteins, which place the pigment granules in their correct
destinations (see Table 3).
One of these categories is particularly topical, as
vesicle trafficking is an area of intense biomedical interest. For example, garnet encodes a ␦-subunit (143), carmine a ␮3-subunit (159), and ruby a ␤-subunit (134) of
the recently discovered AP-3 adaptin complex. It is thus
pleasing to think that a valuable experimental resource
may be provided in the examples above, by direct descendants of flies first isolated in 1916 (33), 1927 (157), and
1910 (C. B. Bridges, unpublished data).
However, there is a further twist: the rekindled interest in vesicle trafficking has focused on the eye phenotype, rather than the tubule. This reflects both the overt
nature of the eye phenotype and the widely held Drosophila perception that only the brain is a respectable alternative topic to development in flies. However, this perception was not always held; the early papers on eye color
mutants invariably sought tubule as well as eye phenotypes. This open-mindedness is entirely laudable, as the
insect eye is a highly specialized and unique tissue; perhaps the more general and physiologically relevant information will be gained from studying vesicle trafficking in
a differentiated, transporting epithelium? Our view of epTABLE
ithelial function increasingly emphasizes the importance
of shuttling differentiated classes of vesicles, carrying
precisely determined cargoes, to specific targets (44, 88,
144, 216). For example, antidiuretic hormone triggers the
translocation of aquaporin-2-containing vesicles to the
plasma membrane of renal cortical collecting duct, a signal that is compromised in diabetes insipidus (232). At
present, much work on membrane trafficking is performed in cell lines (144, 216) that have manifest differences from the differentiated epithelial state. Perhaps the
tubule will be a good place to study some of these translocation and targeting processes. After all, some of the
vesicles are even color-coded.
XII. DROSOPHILA AS A MODEL
FOR HUMAN DISEASE
It is not news that the simple fruitfly, Drosophila
melanogaster, is a useful model for humans, both in
health and disease. However, nearly all Drosophila
groups in the world study developmental biology, and
most of the remainder, brain and behavior. So it is not
surprising to find that the similarities between these very
different organisms (which diverged 400 million years
ago) have been drawn most compellingly in the context of
developmental biology. However, the purpose of this review is to develop an alternative position, that useful
parallels can be drawn in renal function and epithelial
biology. These are areas of great medical significance, but
the similarity of these two organisms has only been hinted
at previously (132).
Despite huge publicity for each success, surprisingly
few genes have been identified as causes of human genetic disease to date. However, a very large fraction of
these genes have homologs in genetic model organisms.
These similarities have been curated (46, 184) and are
available on the internet (http://homophila.sdsc.edu/). [A
similar curation for zebrafish has been published (208).]
Of 929 known human genes with at least 1 mutant allele,
714 (74%) had closely similar matches (P ⬍ 10⫺10 by
BLAST search) in Drosophila. Of these, a useful fraction
3. Some examples of Drosophila eye color mutants for which the loci have been characterized
Gene
Eye Color
Tubule Color
Function
Reference No.
brown
claret
carnation
carmine
cinnabar
deep orange
garnet
Henna
light
lightoid
Brown
Deep reddish yellow
Deep ruby
Carmine
Bright scarlet
Orange
Brownish
Brown to black
Yellowish pink
Clear, light, pink
Slightly paler than wild type
Colorless
Pale yellow
Very pale yellow
Pale yellow
Nearly colorless
Colorless
Wild type to deeper
Colorless
Colorless
ABC transporter for guanine and xanthine
Required for uptake of radiolabeled kynurenin
Vacuolar sorting protein, vps33
Chaperone: clathrin adaptor protein AP3 subunit ␮3
Kynurenine 3-monooxygenase
Vacuolar membrane protein PEP3
Adaptin
Phenylalanine/tryptophan monooxygenase
Vacuolar assembly protein vps41
Likely kynurenine transporter
217
217
21
157
31
26
33
20
56
217
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INTEGRATIVE PHYSIOLOGY AND FUNCTIONAL GENOMICS
had extant mutants: 153 had known alleles, and a further
56 had nearby P-elements that might be useful. Interestingly, although most work in Drosophila has focused on
development, most of the cross-matches in the Homophila database are for metabolic, signaling, or transport enzymes. There is thus ample scope for new work in
Drosophila.
A. Can Drosophila Cure Human Disease?
A critic of the simple model approach for integrative
physiology might argue that no human disease has been
cured by such studies. However, this is a little disingenuous, because the vast majority of funded biomedical
research neither cures nor sets out to cure disease. It is
acknowledged that most research is fundamental, aimed
at understanding the causes of disease, so it would be
unreasonable to apply dual standards. Obviously, it would
not be appropriate (for example) to model the interaction
of vasopressin and the baroreceptor reflex system in the
regulation of arterial blood pressure in a fly, as this is too
narrowly phrased a question. However, there are plenty of
examples of human disease models where insights from
simple organisms are likely to accelerate understanding
or reduce animal use (Table 4).
TABLE
B. How Different Can You Be and Still Be
the Same?
A common criticism of the extension of a comparative physiological approach to invertebrates is the lack of
conservation of body plan. Insects, for example, lack
backbones; they are not mammals; and they lack a developed vascular system. However, all eukaryotic organisms
must accomplish similar functional tasks to stay alive.
The human and Drosophila renal epithelia perform fundamentally analogous tasks, and this review should have
provided sufficient evidence for useful commonality in
function. However, the similarities between disparate
phyla have more spectacular exemplars. For example, the
visual systems of fly and humans are about as dissimilar
as it is possible to be. The former is a faceted eye in which
hundreds of rhabdomeres have distinct but overlapping
visual fields, each lined with multiple photoreceptor cells
in a precisely deterministic pattern (Fig. 24, B and C). The
latter has a single transparent lens, two fluid-filled spaces,
and a complex, multilayered retina. However, there is
remarkable commonality in the genes that specify eye
development. In both fruitfly and human, there are several
single-gene mutations, each capable of blocking eye formation. In humans, these rare syndromes are known as
micropthalmias. Accordingly, these genes are considered
4. Some recent examples of Drosophila models of utility in the study of human disease
Drosophila Model
Human Disease
Hedgehog
Holoprosencephaly (and
others)
ninaD
Blindness through vitamin A
deficiency
Mechanoreception
Deafness
Flies transgenic for
triplet repeats
␣-Synuclein transgenic
flies
Huntington’s disease (and
triplet expansion diseases)
Parkinson’s disease
Flies transgenic for
human tau protein
Technical knockout
Alzheimer’s disease
Bestrophin
Blindness (vitelliform macular
dystrophy)
Linkage disequilibrium
(LD)
Mapping complex human traits
and disease
Deafness
Utility of Model
A signaling pathway of major interest to the fly
community. Accordingly, mutants available in all
points of the pathway: predictive value for
identification of human genes, interactions
NinaD encodes a class B scavenger receptor for
carotenoid uptake. NinaD mutants are blind.
Predicts similar phenotypes for human SR-B1 or
CD36
Aspects of mechanoreception are conserved from
worm to human
Basic understanding, but also allow identification
of modifier loci through suppressor screens
Flies transgenic for ␣-synuclein show neuronal loss
and inclusion bodies, recapitulating human
disease. Transgenic flies also show humanlike
pharmacology. Symptoms are rescued by
overexpression of human or fly hsp70, implying
that chaperones may protect against
neurodegeneration
Flies recapitulate human disease (formation of
neurofibrillary tangles)
Defective in hearing: model for mitochondrial
deafness
Heterologous expression experiments with human
bestrophin identify a new class of oligomeric
chloride channels, and hence the function of the
gene mutated in human disease
The quantitative nature of LD is still being
explored. Combined with SNP analysis, it may
provide a powerful generic tool for mapping
disease loci
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Reference Nos.
15
126
114
42, 103, 122, 195, 210
11,91
112
229
219
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
FIG. 24. Complementation between
mouse and fly eyes absent genes. A: scanning electron micrograph of the side of
the head of an adult eyes absent 2 (eya2)
fly. No facets are visible. B: a normal eye,
formed by ectopic expression of mouse
eya2 gene under UAS control, using a
Drosophila eyeless GAL4 driver. C: electron micrograph of a tangential section
through such an eye, showing that the
stereotyped rhabdomere structure is regained. [From Bonini et al. (28).]
to be high-level developmental switches that trigger eye
development. Remarkably, the genes underlying these
mutations are similar across 400 million years of divergent evolution.
Vertebrate homologs of Drosophila eyes absent
genes are capable of functionally complementing the corresponding Drosophila mutations (Fig. 24), restoring both
morphology and normal function (28). So even in areas of
physiology where there would seem a priori to be little
ground for expecting similarity in function, there is a level
at which the comparison with simple genetic models can
be informative.
C. Functional Genomics Offers a Future
for Comparative Physiology
The utility of genetic models extends phylogenetically in two dimensions. On one hand, the comparison
with humans provides reverse genetics for human functional genomics. On the other hand, simple genetic model
organisms can provide a valuable resource for classical
physiology in closely related species. This can be important where the species is of medical, veterinary, or agricultural significance but lacks the thousands of researcher-years of classical genetic or genomic work that
is the hallmark of a genetic model.
What about physiologists who find that they simply
must work on a target organism that is not a genetic
model? It is still possible to obtain many of the benefits of
the genome project, even in related species. Genome
projects are now spread widely though the Phyla, and it
would be hard to find a species that is not phylogenetically related to a genetic model organism and an associated genome project (Fig. 25). Studies in related species
can then be seen to “cluster” around the models. It is
possible to analyze a particular process in the target
organism, to obtain candidate cDNAs from the phylogenetically closest genome project to use as probes in liPhysiol Rev • VOL
braries from the target organism. Expression patterns can
usefully be mapped by Northerns or in situs in both
species, with the comparative approach providing useful
information. Models of function can then be tested by
reverse genetics in the nearest model, and similarities
inferred with the target organism.
Increasing numbers of species are now being sequenced, beyond the elite list of genetic models in Table
1. This is often for sound economic or medical reasons, as
in the case of the malarial vector Anopheles gambiae or
the Ascidian Ciona intestinalis. Does this weaken the
need for the prototypic, “reference” genetic models as in
Figure 25? On the contrary, it strengthens the case. Reverse genetics is still central to the discovery of function
of novel genes, and although suppression of expression
[e.g., by morpholinos, injection of antisense, or RNA in-
FIG. 25. Relationship between the human genome project, model
organisms, and classical comparative physiology. The acknowledged
importance of reverse genetics has led to the cross-subsidy of genome
projects for a phylogenetically diverse range of organisms.
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INTEGRATIVE PHYSIOLOGY AND FUNCTIONAL GENOMICS
721
terference (79, 80)] can provide some utility in generating
hypomorphs, this is only a partial solution. As has been
illustrated earlier, the depth of genetic knowledge, the
availability of mutants from stock centers, and the powerful reverse genetic technologies add value to the neighboring organisms with recently sequenced genomes. We
illustrate this concept of “model hopping” below, using an
important set of Drosophila’s phylogenetic neighbors:
Dipteran vectors of parasitic disease.
1. Vector biology
Insects make up the large majority of all living species on earth, compared with animals, plants, fungi, or
microbes. The “constituency” of Drosophila thus spans
perhaps 30 million species. In particular, Drosophila is a
thoroughly typical fly, or Dipteran. Many insect pest species are Dipteran, and so are very closely related to Drosophila. Simply by inferring similarity between gene sequences and functions in a given target (pest) species and
Drosophila, it is possible now to draw on many of the
benefits of the Drosophila genome project in advance of
the completion of the Aedes and Anopheles genome
projects.
2. The lower tubule domain is conserved
among Diptera
In the context of the tubule, our understanding of the
Drosophila tubule now outstrips that of any other insect
species. Is it possible to roll out the understanding of
Drosophila to major vectors of parasitic disease, and so
offer new possibilities for selective insecticides? One of
the key insights is the enhancer-trap-derived genetic map
of tubule domains. It would be exciting to establish
whether other Diptera, or other insects, were similarly
organized. However, enhancer trapping depends on transposon mutagenesis and remobilization, and although
these technologies are being pioneered in other Diptera,
they are far from routine (168). A purely morphological
approach is not possible, because there are several regions and cell types in tubules that are morphologically
identical, but which can be distinguished on the basis of
gene expression (Fig. 6). However, enhancer trapping can
lead to the underlying genes, and this in turn can suggest
histochemical, immunocytochemical, or functional assays
that can be applied to other species. As an example, the
two enhancer trap lines that mark the lower tubule domain are insertions in alkaline phosphatase (see sect.
VIIIB8B); and in Drosophila, alkaline phosphatase expression (determined histochemically) maps precisely to the
domain marked out by the relevant enhancer traps (Fig.
26). This provides a simple histochemical assay to test
whether the lower tubule domain is present in nondrosophilid insects. Although the tsetse and mosquito tubules
Physiol Rev • VOL
FIG. 26. The lower tubule domain is conserved among Diptera.
Alkaline phosphatase histochemistry (nitro blue tetrazolium) on adult
tubules of Drosophila and Glossina (A) and Aedes (B). Only the lower
third of the Glossina tubules is shown. (From J. A. T. Dow and S. A.
Davies, unpublished data.)
are 10 times longer, the absolute size of the lower tubule
domain is similar.
In Drosophila, the lower tubule domain marked by
alkaline phosphatase is reabsorptive (see sect. IXA8). Although these pilot data fall short of proof of conserved
function, they suggest interesting and informative experiments.
Another valuable feature of Drosophila has been the
ability to use the GAL4/UAS reporter system to direct
genes of choice to particular genetically defined domains.
In particular, the UAS-aequorin system has allowed cellspecific aspects of neuropeptide signaling to be delineated. In the absence of routine GAL4 enhancer trapping
in other insects, how could this be emulated?
Most insect neuropeptides show some cross-reactivity in other species, although (for example) not all insect
leucokinins stimulate secretion by Drosophila tubules
(63). Once this principle is established for Drosophila
tubules (Fig. 27A), then it is possible to use transgenic
Drosophila tubules, containing stellate cell-directed ae-
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JULIAN A. T. DOW AND SHIREEN A. DAVIES
example with “chip” technologies), there is no doubt that
ultimately a functional analysis of each gene of interest at
an individual level will need to be undertaken. In most
cases, this will demand a reverse genetic approach, and
thus a genetic model. Conversely, the requirement of
integrative physiology to move away from the single molecule, and back to the organism, will often require a set of
transgenic and gene-targeting technologies that overlap
heavily with those of functional genomics. Paradoxically
then, the move away from analytical, molecular, physiology toward a more holistic analysis of organismal function may require a greater level of molecular biological
sophistication than is presently required to perform analytical science.
Although this argument may sound persuasive, it is
not one that has previously been aired extensively. In the
United Kingdom, the Biotechnology and Biological Sciences Research Council mounted two workshops in the
summer of 1998: one each on Functional Genomics and
Integrative Physiology. Significantly, there was almost no
overlap in the delegates!
E. A Plan for Physiologists
From this argument, it is clear that those physiologists who can handle the concept of a genome project
have exciting new opportunities at their disposal.
27. Cross-specificity of neuropeptide action allows for Drosophila bioassays of tsetse peptides. A: secretion of Drosophila tubules
is stimulated by extracts of Glossina central nervous system (CNS). B:
Glossina CNS extract elicits calcium signals in Drosophila tubules and
are measured with transgenic aequorin. (From J. A. T. Dow and S. A.
Davies, unpublished data.)
1. How can genome projects help physiologists?
FIG.
quorin, for example, as cell-specific bioassays for diuretic
or antidiuretic peptides in other species, such as tsetse
(Fig. 27B).
D. Functional Genomics and Integrative Physiology
Are Linked
This line of argument highlights a close linkage between two major areas of contemporary life science, but
one that may not have been perceived. Both functional
genomics and integrative physiology are united in their
dependence on genetic model organisms. (Of course,
even this term is a double-edged sword, as it might imply
wrongly that the organism was of interest only because of
its genetics. We hope that this review will have convinced
the reader otherwise.) Although the emphasis in functional genomics is presently on high-throughput genomics, and then on large-scale target gene selection (for
Physiol Rev • VOL
At its most simple, any physiologist with basic molecular training (or a qualified collaborator) can scrutinize
the sequences output from the genome projects as they
are published, request the DNA clones (they are available
for only a modest charge), sequence, and characterize the
genes.
At the next level, the use of transgenic technologies
afforded by model organisms allows the posing of new
questions in physiology and allows the limitations of cell
culture to be shrugged off with true organotypic transgene expression.
At a further level, by selecting and developing physiological analyses and assays of a particular tissue within
a genetic model, they will be able to harvest the pick of
the novel genes that are already tumbling from the genome projects. In doing so, they not only future-proof
their careers, but provide the bridge between molecular
biology and organismal biology that so many of us feel
needs to be built and strengthened.
2. How can physiologists help genome projects?
For physiologists who can translate their research
into an appropriate model, and so develop a useful phenotype, the genome projects can provide rapid access.
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INTEGRATIVE PHYSIOLOGY AND FUNCTIONAL GENOMICS
For example, a collaboration between SmithKline and the
European Mouse Genome project is presently producing
thousands of mutant mouse lines, with random mutations.
At present, each line is being screened for obvious neurological defects using a 15-min battery of tests designed to
detect inadequacy in a range of indicator behavioral phenotypes (37, 167). However, these mice are equally likely to
have mutations in almost any gene of interest. The collaboration is thus anxious to attract researchers to “hotel” in the
Genome center and screen the mice for any informative
phenotype, while the mice still exist. Simple economics
dictate that only the most interesting lines can be kept alive
for any length of time.
In Drosophila, with its smaller genome size and
lower maintenance costs, the genome project operates
slightly differently. It is possible to contemplate a stock
center holding lethal mutant lines that represent all (or at
least a large fraction) of all essential Drosophila genes. As
a result of some heroic large-scale screening and characterization work, there are now at least 5,000 such lines
available from stock centers. Rather than invoking a “hoteling” approach, these lines are simply made available,
free of charge, to the Drosophila community. So the onus
is on individual experimenters to realize that they need a
particular line by virtue of its documented phenotype.
Other genome projects can be expected to operate
somewhere on this continuum, based on the trade-off
between genome size, maintenance cost, and ease of mutagenesis of the genetic model organism.
XIII. FUTURE PROSPECTS
Our analysis suggests a bright future for functional
(physiological) research, with benefits both to the physiologist and to the genomics communities. However, there
is a deeper message: that physiologists, rather than agonizing over whether molecular biology is a friend or foe,
must set their horizons very much wider. The linkage we
have demonstrated between functional genomics and integrative physiology is just an example of the integration
of the life sciences themselves. Physiology, genetics, and
molecular biology are combinations of philosophies and
techniques that are convergent and will become unified in
the postgenomic “new biology.”
XIV. CONCLUSIONS
Integrative physiology and functional genomics converge on their need for genetic models, transgenics, and
physiology. The Drosophila tubule demonstrates the usefulness of genetic technology to physiology. Drosophila is
an ideal model for study of human diseases, and not just
of development or brain.
Physiol Rev • VOL
723
We thank the referees for their helpful comments.
This work was supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust.
Address for reprint requests and other correspondence:
J. A. T. Dow, Div. of Molecular Genetics, Institute of Biomedical
and Life Sciences, Univ. of Glasgow, Glasgow G11 6NU, UK
(E-mail: j.a.t.dow@bio.gla.ac.uk).
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