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First published online April 20, 2007
Journal of Experimental Biology 210, 1632-1640 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.002691

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Review Article

Integrative physiology, functional genomics and the phenotype gap: a guide for comparative physiologists

Julian A. T. Dow

Division of Molecular Genetics, University of Glasgow, Glasgow, G11 6NU, UK

e-mail: j.a.t.dow{at}bio.gla.ac.uk

Accepted 23 January 2007

	Summary

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
Classical, curiosity-led comparative physiology finds itself at a crossroads. Major funding for classical physiology is becoming harder to find, as grant agencies focus on more molecular approaches or on science with more immediate strategic value to their respective countries. In turn, this shift in funding places Zoology and Animal Science departments under enormous stress: student numbers are buoyant, but how can research funding be maintained at high levels?

Our research group has argued for the redefinition of integrative physiology as the investigation of gene function in an organotypic context in the intact animal. Implicit in this definition is the use of transgenics and reverse genetics to manipulate gene function in a cell-specific manner; this in turn implies the use of a genetically tractable `model organism'. The significance of this definition is that it aligns integrative physiology with functional genomics. Again, functional genomics draws heavily on reverse genetics to elucidate the function of novel genes. The phenotype gap (the mismatch between what a genetic model organism's genome encodes and the reasons that it has historically been studied) emphasises the need to attract and empower functional biologists: can all 13 500 genes in Drosophila really be explained in terms of developmental biology? So, by embracing the integrative physiology manifesto, comparative physiologists can not only accelerate their own research, but their functional skills can make them indispensable in the post-genomic endeavour.

Key words: Drosophila melanogaster, Caenorhabditis elegans, Danio rerio, mouse, Malpighian tubule, bioinformatics

	Introduction

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
This paper is aimed at students and academics who are contemplating whether to attempt real, integrative physiology – particularly in the fruit fly, Drosophila melanogaster. It sets out the reasons why this may be desirable and outlines the basic steps needed to obtain the resources required and to start experimenting.

	Integrative physiology and functional genomics

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
Physiology is the study of how a living organism works. Classical physiologists are becoming an endangered species, just as the need for their skills is growing once again. This shortfall is for several reasons, not least the stringency of regulations for animal work but also because non-molecular work is now discouraged by many grant agencies. Nonetheless, cellular and molecular approaches to physiology have proved potent and cost-effective paradigms. Ultimately, however, these reductionist or analytical methodologies can prove restricting: how can we draw on the strengths of these methodologies, while addressing function in the whole organism – the new, `integrative physiology'?

Our group has argued that, rather than abandoning molecular biology in a return to classical techniques, it is possible to integrate a gene-based approach into studies of the whole organism (Dow and Davies, 2003). Integrative physiology is seen – as in the present motto of the American Physiological Society – as the move `from gene to tissue to organism'. How can this be achieved? We have argued that transgenic organisms, in which expression of a particular gene of interest is manipulated upward or downward in a cell-specific manner, provide a potent link between gene and organism (Dow and Davies, 2003). In essence, physiologists are exhorted to adopt the skills of the geneticist, by trying to understand the function of specific genes by disrupting them or modulating them – what is known as `reverse genetics'. This philosophy is not merely academic: it was adopted by the UK's Biotechnology and Biological Sciences Research Council in their recent `Genomics in Animal Function (GAIN)' Initiative.

The attraction of this working definition of integrative physiologist is that it aligns comparative physiology with one of the most important research fields today; that of functional genomics – the understanding of the function of all the genes encoded in a genome. Perhaps one-third of the genes in any genome (so perhaps 5000–10 000) are sufficiently novel that their function cannot be predicted in silico; for such genes, a reverse genetic work-up is considered one of the most powerful approaches.

For functional genomics, however, there is a major log jam in the reverse genetic pipeline; there is no point in mutating a particular gene unless the experimenter is able to recognize and study any resulting phenotype. Functional genomics thus requires the broadest range of functional biologists to align themselves with their endeavour. The problem is that model organisms have come to prominence, not for their physiological tractability but for highly focused studies – typically of development. The `phenotype gap' is therefore the mismatch between the functions encoded by an organism's genome and what it has historically been used to study (Brown and Peters, 1996; Bullard, 2001; Dow, 2003; Wang et al., 2004). The scale of this mismatch for most model organisms emphasizes the opportunities available to comparative physiologists flexible enough to take on a new organism.

It is actually possible to quantify the phenotype gap. By the time the Drosophila genome was sequenced in 2000, I estimate that at least 300 000 researcher-years had been devoted to Drosophila – the large majority spent on studies of development. Drosophila genes are given (more or less witty) names as they are first encountered, and so it is possible to distinguish known, studied genes from those that are merely computer annotated. Of the 13 500 or so genes identified in the first release of the genomic sequence, only 20% were named (Wang et al., 2004). We can thus assume that developmental biology, as a screen for new genes, has now neared saturation. For the other 80% of genes, we need to seek new phenotypes.

	The need for model organisms

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
Once the case for transgenics as a physiological tool is accepted, then the choice of organism is immediately circumscribed. Transgenics is only feasible in the small set of organisms (like mice, fruit flies, worms or zebra fish) known as genetic models (Table 1). Typically, these organisms have fully sequenced genomes and a wealth of freely available mutant stocks, or the resources to create more as required. However, this appears to run contrary to the Krogh principle, beloved of comparative physiologists; that for any physiological question, there is a species in which it can best be studied (Krogh, 1929). When there are perhaps 30 million species of animal (mostly insect) in the world, how can it be sensible to restrict oneself to less than a few tens of species?

The answer is twofold. Firstly, integrative physiology is not doctrinaire; it remains possible to continue working in a target species, while dipping occasionally into the remarkable resources associated with the model organism. For example, an insect physiologist working on an agricultural pest organism could look up the sequence of a particular neuropeptide receptor from the Drosophila genome project and use it to design primers that would be likely to pull the gene out of the target organism. Alternatively, if a gene was identified by an advanced molecular technique like suppression subtractive hybridization in the target species, mutants could be sought in the phylogenetically closest model species, and studied there, so as to understand the gene of interest rather better. The second answer is that, just because a model organism has a sequenced genome and a wealth of genetic resources, it is not intrinsically less interesting than a non-model; indeed, if one considers that the Krogh principle applies both to an organism that exemplifies a trait `and the ease with which it can be studied experimentally', then model organisms can acquire Krogh status for a surprising range of studies.

	Real physiology

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
So far, these arguments could be seen as philosophical. However, taking Drosophila as an example, it is possible to identify several examples of real physiology interacting closely with genetics in order to provide powerful, fundamental insights.

1. Our understanding of the circadian clock is based on pioneering (and painstaking) screens of insects that failed to eclose from their pupae at the normal time of day, implying that they had lost track of time (Konopka and Benzer, 1971). Mutants discovered in Drosophila, like period and timeless, have influenced the whole field.
   
2. Similarly, mutants identified in simple learning paradigms have implicated the cyclic AMP signalling pathway; dunce is a cAMP phosphodiesterase, while rutabaga is an adenylate cyclase (Dudai et al., 1976; Dudai and Zvi, 1985). The work in Drosophila thus closely aligns with Kandel's Nobel prize-winning studies on habituation of the gill withdrawal reflex in the non-model sea hare, Aplysia californica (Kandel and Schwartz, 1982).
   
3. The suspicion that the 24-transmembrane pass voltagegated ion channels were actually derived from two gene duplication events acting on a 6-transmembrane pass ancestor was dramatically confirmed when a line of flies with legs that continued to shake under ether anaesthesia (Catsch, 1944) were shown to be mutants in a potassium channel gene, named Shaker (Kamb et al., 1987). At first sight, one might think it only appropriate that a primitive channel had been identified in a `primitive' organism, but this would show a grave misunderstanding of evolution. Drosophila has continued to evolve in the 400 million years since it diverged from the common ancestor of humans, so it is different, rather than primitive. Indeed, the Shaker channel of Drosophila triggered the discovery of a ubiquitous family of quarter-sized channels that were found even in humans (Salkoff et al., 1992).
   

Of course, these physiological examples are all drawn from neuroscience. Our group's work, however, has shown that renal function can also be studied to great advantage in Drosophila (Dow and Davies, 2001; Dow and Davies, 2003; Dow and Davies, 2006). The sequenced genome allowed the rapid identification of genes encoding diuretic neuropeptides (Cabrero et al., 2002; Coast et al., 2001; Kean et al., 2002; Terhzaz et al., 1999) and their receptors (Johnson et al., 2005; Radford et al., 2002), often before it proved possible in nonmodel organisms, and indeed these studies paved the way for similar work in other insects (Radford et al., 2004).

	Using the tools

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
The key genetic tool for Drosophila physiology is the GAL4 enhancer trap. This has been described in detail many times (Brand and Perrimon, 1993; Sentry et al., 1994) but, in essence, it provides the ability to express genetic constructs of choice in specific cells in an otherwise normal organism – exactly the technological requirements for integrative physiology (Dow and Davies, 2003). Targeted ectopic expression is possible in a wide range of genetic models, but perhaps nowhere as simply as in Drosophila. The GAL4/UAS system is binary; that is, a fly is generated by crossing together a `driver' line (one in which the yeast transcription factor GAL4 is expressed in a desired pattern) and a UAS line (one in which the genetic payload is placed downstream of five copies of the UAS binding site recognized by GAL4). In such flies, the genetic payload is switched on strongly in those cells in which GAL4 is being expressed (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1. The GAL4/UAS system. Modified from Dow (Dow, 2007).

However, there is still greater sophistication available. What if it is desirable to express a gene of interest not just in a particular spatial pattern but at a particular time? For example, it might be important to express a deleterious construct only in adult Drosophila just before study, to prevent pleiotropic effects (or even lethality) in the embryo or larva. This can be achieved with a temperature-sensitive GAL4-binding protein, GAL80. In flies transgenic for a GAL4 driver, a UAS-driven transgene and GAL80, GAL4 is expressed in a spatially restricted pattern, but bound by GAL80, thus preventing it from binding to UAS and activating the transgene. However, when the temperature is raised to 30°C, GAL80 dissociates, allowing GAL4 to bind to UAS and so activate the transgene (McGuire et al., 2004; Suster et al., 2004).

Another strategy is to provide the genetic equivalent of a `latching' switch for the GAL4/UAS system. If a fly is generated containing a GAL4 driver and a UAS-driven payload, but in the additional presence of a GAL4 transgene downstream of a UAS promoter, then wherever GAL4 is transiently expressed, it will activate the GAL4 transgene, so providing high levels of GAL4 expression indefinitely in that cell (Hassan et al., 2000).

Sometimes, it can be hard to study the effects of a gene because available mutants are lethal. The traditional Drosophila genetic technique is to generate X-ray-induced mosaics, in which the mutation is only homozygous in a small population of cells (Becker, 1975). Clever experimental design can allow these cells to be visibly marked; these mutant patches can then be studied in the context of an otherwise normal animal. Yeast technology adds a modern twist to these classical experiments; if the lethal mutation is crossed onto a chromosome that has a yeast flippase recombination target (FRT) site near its centromere, and then yeast flippase (FLP) is driven transgenically, there is a finite chance that the chromosomes will recombine (Golic and Lindquist, 1989; Xu and Harrison, 1994). This has the effect of producing clones of cells carrying two mutant (or two wild-type) chromosomes. Of course, if UAS–FLP is driven with GAL4, it is possible to target specific populations of cells, rather than throughout the organism.

RNA interference (RNAi) by double-stranded RNA, originally employed in Caenorhabditis elegans (Fire et al., 1998), has proved a potent means of obtaining hypomorphic alleles of genes, without resorting to screening for new mutants. Although many means of expressing double-stranded RNA (dsRNA), the precursor of short interfering RNA (siRNA), have been developed, the favoured means at present is to use Carthew's pWiz vector (Lee and Carthew, 2003). This has a complete pUC plasmid with antibiotic resistance marker (so that it can be grown up in Escherichia coli), P-element ends (so that it can transform into Drosophila at high efficiency), a white minigene (to allow transgenic flies to be identified by their red eyes) and rare-cutter sites (downstream of UAS) that allow DNA fragments to be dropped into the vector in opposite orientations (Fig. 2). The cloning sites are on either side of an efficiently spliced white intron, believed to help in the generation of dsRNA. This makes the generation of flies transgenic for UAS–RNAi constructs a matter of a week's cloning, followed by a three-month period of intermittent fly husbandry.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Schematic of the pWiz vector. Reproduced with permission from Lee and Carthew (Lee and Carthew, 2003).

The weakness of many model organisms is the difficulty or impossibility of targeted homologous recombination. Rather than just adding a transgene, this is the ability to replace a particular genomic sequence with another sequence; for example, to replace the open reading frame of a gene with green fluorescent protein (GFP), so inactivating the gene while putting the reporter under precisely the combination of promoters and enhancers that control the wild-type gene. In Drosophila, this is now possible, although not trivial. Golic's lab realized that the limitation was in producing a linear targeting construct capable of recombination with the Drosophila germ-line (Rong and Golic, 2000; Rong et al., 2002). The procedure they designed involves inserting the targeting construct stably into the germline by conventional transformation, then excising it at FRT sites with a transgenic FLP recombinase enzyme. The targeting construct is then able to synapse with its target, allowing recombination to take place with reasonable efficiency (Rong and Golic, 2000; Rong et al., 2002).

The jewel in the crown of Drosophila genetics, however, is its long history of community-generated resources. The largest stock centre, in Bloomington (http://flystocks.bio.indiana.edu/), holds over 20 000 mutant fly lines, available for a nominal charge by e-mail: requests are processed within a couple of weeks. Not only does this imply that there is a good chance that an informative allele for a particular gene may already exist but there is also the possibility of handling the direct descendents of flies identified by the Drosophila pioneers, Morgan, Bridges and Sturtevant. For example, we were recently able to document the recapitulation of the human renal disease xanthinuria type I in mutants at the Drosophila rosy locus, using the direct descendents of the rosy² mutants first identified around 1916 (Wang et al., 2004). The utility of these mutants is increased by the ease with which they can be identified; the freely accessible Flybase website (http://www.flybase.net/) is exemplary in its ease of use.

Of course, similar results could be achieved in other organisms; it is the speed and cost that makes the Drosophila version of the technology so attractive.

	Drosophila and other models

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
I suggest that there are certain minimum requirements on a model organism for it to be useful for integrative physiology. It must be possible to introduce transgenes, to produce mutations of specific genes and to intervene in a tissue-specific manner. It must also be possible to perform physiology on the organism and it is highly desirable that there should be a sequenced genome, as this makes many aspects of the work far easier.

In this context, it becomes clear that the trade-off between biomedical relevance and genetic power does not automatically lead to the mouse as uniformly best choice (Table 1).

Put simply, a transgenic mouse line takes several years, and perhaps in excess of $100 000 to realize, and then $10 000/year to maintain thereafter; whereas a transgenic fruit-fly line can be made for $500 in three months and then costs $30/year to maintain. Clearly, an investigator can gain time and flexibility, while saving considerable amounts of money, if their questions can be addressed in a simpler model.

Neither should it be assumed that biomedical research is the only kind worth doing! Integrative physiology has perhaps as many as 30 million species with which to work; and the demands of both basic, curiosity-led research and perceived `usefulness' do not have to be antagonistic. For example, many millions of lives are lost annually to parasitic diseases (notably, but not exclusively, malaria) that are carried by Diptera – phylogenetically close relatives of Drosophila (Butler, 2003).

More generally, is there thus an adequate phylogenetic spread of model species such that integrative biology can be evoked for a good proportion of problems in comparative physiology? So far, the match is not perfect, according to the NCBI's genome page (http://www.ncbi.nlm.nih.gov/Genomes/). Mammals are well-represented for genome projects, and mouse is the mainstay model organism. The position is similar for insects, with Drosophila melanogaster as the constituency champion (and sequencing underway or complete for a total of 50 species). For birds, the chicken genome is now released, but the transgenic resources are not yet as potent as for the first models. For this reason, much is made of a particularly easy-to-transform lymphoid cell line with high rates of recombination, DT40 (Buerstedde et al., 1990) – although this of course is hardly `integrative' as we have defined it. Other species, such as the zebra finch, Taeniopygia guttata, are being sequenced. For fish, fugu (Takifugu rubripes), the related pufferfish Tetraodon nigroviridis and zebra fish (Danio rerio), and Oryzias latipes (Japanese medaka) sequences are nearly complete; but targeted mutagenesis has been announced several times, rather than been deployed as a routine tool, in fish. Elasmobranchs are represented by Leucoraja erinacea (little skate). The Reptilia are conspicuously unsequenced, and the Amphibia are represented by Xenopus tropicalis (western clawed frog).

Among the simpler animal phyla, the nematode worm C. elegans has remarkably potent genetic tools, especially for making transgenics; it is possible to microinject embryos with plasmids that then replicate as episomal `rafts', so providing effectively stable transformants overnight. By generating worms transgenic for fluorescent reporters, mutagenising them and passing them through essentially a modified FACS (fluorescence activated cell sorting) machine, it has proved possible to screen 300 000 worms in a weekend – a throughput which is the envy of the fly community (Strange, 2003; Strange, 2007). As well as several other members of the genus Caenorhabditis, the nematode roundworm Trichinella spiralis (the cause of human trichinosis) is being sequenced.

Outside of these `hotspots', however, things get patchier. The echinoderm species Strongylocentrotus purpuratus is being sequenced, and there is an expressed sequence tag (EST) sequencing project underway for the crab Carcinus maenus, and a genome project for the water flea Daphnia pulex, the tick Ixodes scapularis, the hemichordate acorn worm Saccoglossus kowalevskii, the freshwater planarian Schmidtea mediterranea and the pig tapeworm Taenia solium. For the molluscs, sequencing of the Atlantic surf clam (Spisula solidissima) and Biomphalaria glabrata (the freshwater snail host for schistosomiasis) is underway, and the sequence for Aplysia californica (California sea hare) is being assembled. In the even simpler cnidarians, assembled genomic sequence is available for two sea squirts (Cionia spp.), and a sea anemone (Nematostella vectensis) and the hydrozoan polyp Hydra magnipapillata are being sequenced. Perhaps most exotically, the tunicate Oikopleura dioica and the simplest known animal (and only known member of the Placozoa), Trichoplax adhaerens, are being sequenced. These data are summarized in Table 2.

Of course, genomic sequence is only one criterion for integrative biology, and it must be conceded that there are no publications describing transgenic technologies for the large majority of the organisms listed above. Nor can many of the species listed above be considered to be established physiological models. So, the list of models compatible with integrative physiology in Table 1 remains fairly definitive for the time being, until the genetics catches up with the genomics.

	The future for model organisms

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
If one considered genomic sequence and transgenic technology to be sufficient for integrative biology, then the days of the models might be limited. For example, now that genomic sequence is available for the malaria mosquito Anopheles gambiae and the yellow fever mosquito Aedes aegypti, and germ line transformation with transposon-based vectors has shown to be feasible for both, has Drosophila had its day? Interestingly, perhaps the opposite holds true; comparative genomics approaches make it much easier to drill through to a model such as Drosophila and to access the extant mutant stocks and long-term research database available for the model species. The Bloomington stock centre alone holds over 20 000 stocks; whereas the technical difficulty of keeping mosquitoes means that even an active lab could not hope to maintain more than perhaps six – and then only while grant funding continued. So the `model organism package' needs to be seen as a whole.

	RNAi – does it promote `target' species to `model' status?

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
If the utility of mutants is accepted as a key property of a model, can RNAi be used to provide mutants in non-model species? Since its discovery, RNAi has proved a powerful experimental tool, particularly in C. elegans, where it suffices to feed worms on E. coli harbouring a plasmid encoding an RNAi hairpin construct (Wang and Barr, 2005). In Drosophila, there are transformation vectors available that make generation of dsRNA particularly easy (Fig. 2), and there are genome-wide screens underway both in whole flies and cell lines.

However, problems remain; RNAi relies on the subversion of a mechanism probably intended to attack invading viruses, and a common side-effect of RNAi treatment is the nonspecific shutdown of transcription. Care must be taken to control also for the knockdown of closely related transcripts (so-called `off-target effects'). In addition, most RNAi alleles are hypomorphs, rather than nulls. This can be an advantage, as lethal mutations can be hard to study! However, we find that driving high levels of dsRNA inside cells with the GAL4/UAS system still only produces detectable knockdowns in about half of all cases (J.A.T.D., unpublished).

Given that RNAi is only partially effective when actually generated inside a cell, this might make one sceptical of the miraculous properties ascribed to RNAi in non-model organisms. dsRNA has been applied in the food, or injected into the haemocoel of larval and adult insects, and found to produce complete knockdowns that last for days, and in some cases have been heritable. These results imply that dsRNA is able to tunnel across basement membranes as well as plasma membranes, and sometimes even the blood–brain barrier. So, although RNAi (or morpholinos) will prove important in nonmodel species, it must be seen as a maturing technology that requires strict controls to be respected. For example, western blotting with a specific antibody to show protein knockdown, accompanied by counterstaining for a related protein that is not affected, should be seen as a minimum requirement.

	Diversity and the insects

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
Model organisms are vital tools, but do they actually have any relevance for target species? More generally, do data in any given species have relevance to other species? This question is particularly acute for the insects, where there are perhaps as many as 30 million species in existence. Is it possible to estimate the extent to which Drosophila is a model insect, rather than a model fly? And is it possible to design a rational sampling strategy to cover this wide diversity with finite resources?

In the case of the Drosophila tubule, the evidence is promising. The importance of the apical V-ATPase is known from many species (Bertram et al., 1991; Dow et al., 1994; Garayoa et al., 1995; Maddrell and O'Donnell, 1992; Pietrantonio and Gill, 1995), and the indications that a basolateral, glibenclamide and barium-sensitive potassium channel is important are also well known (Beyenbach and Masia, 2002; Evans et al., 2005; Masia et al., 2000; Weltens et al., 1992; Wiehart et al., 2003a).

Neuroendocrine control shows great commonality, implying that neuropeptides have a common origin in insects. In all insects studied so far, cyclic AMP is diuretic and can be raised by either the corticotropin releasing factor-like DH₄₄ or the calcitonin-like DH₃₁ (Coast, 1998). Although the similarities with the vertebrate peptides are very faint, it could be argued that signalling through these peptides is conserved beyond insects.

The case can be made much more strongly, however, for the leucokinin family. Indeed, although the first leucokinins were characterized in an insect (Holman et al., 1984), the first gene for a leucokinin receptor, and its cognate peptide, were identified in the pond snail Lymnaea stagnalis in a single, thoroughly impressive paper (Cox et al., 1997). Similarly, leucokinin signalling is known to occur in the Acari (mites and ticks) (Holmes et al., 2000). In insects, leucokinins are uniformly myogenic and diuretic (Coast et al., 2002). Drosophila has proved useful in elucidating the mode of action of these neuropeptides; not only were the first insect leucokinin and leucokinin receptor genes identified in Drosophila, but the mode of action (through intracellular calcium) was established with transgenic calcium reporter technology (Radford et al., 2002; Terhzaz et al., 1999). Leucokinin signalling is sufficiently well conserved that neuropeptides can be active across wide phylogenetic distances in the insects.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Reconciling tubule physiology with phylogeny. Five species are represented; the muscomorph flies Drosophila melanogaster and the tsetse fly Glossina morsitans; the mosquitoes Aedes aegypti and Anopheles gambiae; and the more primitive orthopteran Schistocerca gregaria. Some recent comparative results from our lab are overlaid with classical data; the number of tubules per animal (N=xx), the approximate number of cells per tubule (brown shading), the presence of nitric oxide synthase in the tubule (NOS) (Pollock et al., 2004), the diuretic activity of Capa (CAPA) (Pollock et al., 2004), the existence of a defined alkaline phosphatase domain in the lower (proximal) tubule (ALP) (Cabrero et al., 2004), the presence of stellate cells (green stars) (Cabrero et al., 2004) and the haematophagous habit (red drops). The diagram shows that some properties can be considered to be common to insects whereas others seem to be associated with the Diptera. Conspicuously, tsetse flies, although closely related to Drosophila, lack stellate cells: parsimony suggests this is a secondary loss, perhaps associated with the degenerate lifestyle of these unusual flies.

So, overall, it looks as if data from Drosophila Malpighian tubules rolls out across all Diptera with very few caveats, and indeed the broad pattern of tubule function and control is recognizable across all insects (Fig. 3). So, the model organism/integrative physiology agenda seems to survive this relatively severe test (up to 30 million species in over 20 orders separated by 150 million years of rapid divergent evolution).

	Conclusion

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 
This review has shown that there is considerable promise in the judicious use of model organisms to establish general principles of function. It should be clear that the ease and power with which some of these organisms can be manipulated does elevate them to `Krogh status' for several interesting biological questions. The Drosophila Malpighian tubule is in no way an exceptional tissue in an exotic organism; it has simply been studied in some detail. Clearly, it is vital for functional genomics that more experimental biologists take an active interest in specific tissues in these valuable organisms. This will lay the ground not just for a better understanding of what genes do but for a systems approach to the function of the whole organism. Surely this should be the ultimate goal of integrative physiology?

	Acknowledgments

 
Our group is generously supported by the UK's Biotechnology and Biological Sciences Research Council (BBSRC). I am most grateful to Dr Shireen Davies for her critical reading of the manuscript.

	Footnotes

 
Glossary available online at http://jeb.biologists.org/cgi/content/full/210/9/1632/DC1

	References

TOP Summary Introduction Integrative physiology and... The need for model... Real physiology Using the tools Drosophila and other models The future for model... RNAi - does it... Diversity and the insects Conclusion References

 

Becker, H. J. (1975). X-ray- and TEM-induced mitotic recombination in Drosophila melanogaster: unequal and sister-strand recombination. Mol. Gen. Genet. 138, 11-24.[CrossRef][Medline]

Bertram, G., Shleithoff, L., Zimmermann, P. and Wessing, A. (1991). Bafilomycin_A1 is a potent inhibitor of urine formation by Malpighian tubules of Drosophila hydei – is a vacuolar-type ATPase involved in ion and fluid secretion? J. Insect Physiol. 37,201 -209.[CrossRef]

Beyenbach, K. W. and Masia, R. (2002). Membrane conductances of principal cells in Malpighian tubules of Aedes aegypti.J. Insect Physiol. 48,375 -386.[CrossRef][Medline]

Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118,401 -415.[Abstract]

Brown, S. D. M. and Peters, J. (1996). Combining mutagenesis and genomics in the mouse – closing the phenotype gap. Trends Genet. 12,433 -435.[CrossRef][Medline]

Buerstedde, J. M., Reynaud, C. A., Humphries, E. H., Olson, W., Ewert, D. L. and Weill, J. C. (1990). Light chain gene conversion continues at high rate in an ALV-induced cell line. EMBO J. 9,921 -927.[Medline]

Bullard, D. C. (2001). Mind the phenotype gap. Trends Mol. Med. 7,537 -538.[CrossRef][Medline]

Butler, D. (2003). Gates steps up war on malaria with donation of 168 million dollars. Nature 425, 331.[Medline]

Cabrero, P., Radford, J. C., Broderick, K. E., Costes, L., Veenstra, J. A., Spana, E. P., Davies, S. A. and Dow, J. A. T. (2002). The Dh gene of Drosophila melanogaster encodes a diuretic peptide that acts through cyclic AMP. J. Exp. Biol. 205,3799 -3807.[Abstract/Free Full Text]

Cabrero, P., Pollock, V. P., Davies, S. A. and Dow, J. A. T. (2004). A conserved domain of alkaline phosphatase expression in the Malpighian tubules of dipteran insects. J. Exp. Biol. 207,3299 -3305.[Abstract/Free Full Text]

Catsch, A. (1944). Eine erbliche Storung des Bewegungsmechanismus bei Drosophila melanogaster. Z. Indukt. Abstamm. Verebungsl. 82,64 -66.

Coast, G. M. (1998). Insect diuretic peptides: structures, evolution and actions. Am. Zool. 38,442 -449.

Coast, G. M., Webster, S. G., Schegg, K. M., Tobe, S. S. and Schooley, D. A. (2001). The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J. Exp. Biol. 204,1795 -1804.[Abstract]

Coast, G. M., Orchard, I., Phillips, J. E. and Schooley, D. A. (2002). Insect diuretic and antidiuretic hormones. Adv. Insect Physiol. 29,279 -409.[CrossRef]

Cox, K. J., Tensen, C. P., Van der Schors, R. C., Li, K. W., van Heerikhuizen, H., Vreugdenhil, E., Geraerts, W. P. and Burke, J. F. (1997). Cloning, characterization, and expression of a G-protein-coupled receptor from Lymnaea stagnalis and identification of a leucokinin-like peptide, PSFHSWSamide, as its endogenous ligand. J. Neurosci. 17,1197 -1205.[Abstract/Free Full Text]

Davies, S. A., Dow, J. A. T., Huesmann, G. R., Maddrell, S. H. P., O'Donnell, M. J. and Tublitz, N. J. (1994). CAP_2b, a cardioactive peptide of lepidopteran insects, can also stimulate fluid production by Malpighian tubules of Drosophila melanogaster through a mechanism involving cGMP. J. Physiol. 482,26P .

Davies, S. A., Stewart, E. J., Huesmann, G. R., Skaer, N. J. V., Maddrell, S. H. P., Tublitz, N. J. and Dow, J. A. T. (1997). Neuropeptide stimulation of the nitric oxide signaling pathway in Drosophila melanogaster Malpighian tubules. Am. J. Physiol. 42,R823 -R827.

Dow, J. A. (2003). The Drosophila phenotype gap – and how to close it. Brief. Funct. Genomic. Proteomic. 2,121 -127.[Abstract/Free Full Text]

Dow, J. A. T. (2007). Model organisms and molecular genetics for endocrinology. Gen. Comp. Endocrinol. doi:10.1016/j.ygcen.2007.01.023 .

Dow, J. A. T. and Davies, S. A. (2001). The Drosophila melanogaster Malpighian tubule. Adv. Insect Physiol. 28,1 -83.

Dow, J. A. T. and Davies, S. A. (2003). Integrative physiology and functional genomics of epithelial function in a genetic model organism. Physiol. Rev. 83,687 -729.[Abstract/Free Full Text]

Dow, J. A. T. and Davies, S. A. (2006). The Malpighian tubule: rapid insights from post-genomic biology. J. Insect Physiol. 52,365 -378.[CrossRef][Medline]

Dow, J. A. T., Maddrell, S. H. P., Görtz, A., Skaer, N. V., Brogan, S. and Kaiser, K. (1994). The Malpighian tubules of Drosophila melanogaster: a novel phenotype for studies of fluid secretion and its control. J. Exp. Biol. 197,421 -428.[Abstract]

Dudai, Y. and Zvi, S. (1985). Multiple defects in the activity of adenylate cyclase from the Drosophila memory mutant rutabaga. J. Neurochem. 45,355 -364.[CrossRef][Medline]

Dudai, Y., Jan, Y. N., Byers, D., Quinn, W. G. and Benzer, S. (1976). dunce, a mutant of Drosophila deficient in learning. Proc. Natl. Acad. Sci. USA 73,1684 -1688.[Abstract/Free Full Text]

Evans, J. M., Allan, A. K., Davies, S. A. and Dow, J. A. T. (2005). Sulphonylurea sensitivity and enriched expression implicate inward rectifier K⁺ channels in Drosophila melanogaster renal function. J. Exp. Biol. 208,3771 -3783.[Abstract/Free Full Text]

Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature 391,806 -811.[CrossRef][Medline]

Garayoa, M., Villaro, A. C., Klein, U., Zimmermann, B., Montuenga, L. M. and Sesma, P. (1995). Immunocytochemical localization of a vacuolar-type ATPase in Malpighian tubules of the ant Formica polyctena. Cell Tissue Res. 282,343 -350.[CrossRef]

Golic, K. G. and Lindquist, S. (1989). The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59,499 -509.[CrossRef][Medline]

Hassan, B. A., Bermingham, N. A., He, Y., Sun, Y., Jan, Y. N., Zoghbi, H. Y. and Bellen, H. J. (2000). atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain. Neuron 25,549 -561.[CrossRef][Medline]

Holman, G. M., Cook, B. J. and Wagner, R. M. (1984). Isolation and partial characterization of five myotropic peptides present in head extracts of the cockroach, Leucophaea maderae.Comp. Biochem. Physiol. 77C,1 -5.[CrossRef][Medline]

Holmes, S., He, H., Chen, A., Ivie, G. and Pietrantonio, P. (2000). Cloning and transcriptional expression of a leucokinin-like peptide receptor from the southern cattle tick, Boophilus microplus (Acari: Ixodidae). Insect Mol. Biol. 9, 457-465.[CrossRef][Medline]

Johnson, E. C., Shafer, O. T., Trigg, J. S., Park, J., Schooley, D. A., Dow, J. A. T. and Taghert, P. H. (2005). A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. J. Exp. Biol. 208,1239 -1246.[Abstract/Free Full Text]

Kaiser, K. (1993). Transgenic Drosophila – second generation enhancer traps. Curr. Biol. 3,560 -562.[CrossRef][Medline]

Kamb, A., Iverson, L. E. and Tanouye, M. A. (1987). Molecular characterization of Shaker, a Drosophila gene that encodes a potassium channel. Cell 50,405 -413.[CrossRef][Medline]

Kandel, E. R. and Schwartz, J. H. (1982). Molecular biology of learning: modulation of transmitter release. Science 218,433 -443.[Abstract/Free Full Text]

Kean, L., Pollock, V. P., Broderick, K. E., Davies, S. A., Veenstra, J. and Dow, J. A. T. (2002). Two new members of the CAP_2b family of diuretic peptides are encoded by the gene capability in Drosophila melanogaster. Am. J. Physiol. 282,R1297 -R1307.

Konopka, R. J. and Benzer, S. (1971). Clock mutants of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 68,2112 -2116.[Abstract/Free Full Text]

Krogh, A. (1929). The progress of physiology. Am. J. Physiol. 90,243 -251.[Free Full Text]

Lee, Y. S. and Carthew, R. W. (2003). Making a better RNAi vector for Drosophila: use of intron spacers. Methods 30,322 -329.[CrossRef][Medline]

MacPherson, M. R., Lohmann, S. M. and Davies, S. A. (2004). Analysis of Drosophila cGMP-dependent protein kinases and assessment of their in vivo roles by targeted expression in a renal transporting epithelium. J. Biol. Chem. 279,40026 -40034.[Abstract/Free Full Text]

Maddrell, S. H. P. and O'Donnell, M. J. (1992). Insect Malpighian tubules: V-ATPase action in ion and fluid transport. J. Exp. Biol. 172,417 -429.[Abstract/Free Full Text]

Masia, R., Aneshansley, D., Nagel, W., Nachman, R. J. and Beyenbach, K. W. (2000). Voltage clamping single cells in intact Malpighian tubules of mosquitoes. Am. J. Physiol. Renal Physiol. 279,F747 -F754.[Abstract/Free Full Text]

McGuire, S. E., Mao, Z. and Davis, R. L. (2004). Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila. Sci. STKE 2004, pl6.[Abstract/Free Full Text]

Pietrantonio, P. V. and Gill, S. S. (1995). Immunolocalization of the 17-Kda vacuolar H⁺-atpase subunit-C in Heliothis virescens midgut and Malpighian tubules with an antipeptide antibody. J. Exp. Biol. 198,2609 -2618.[Medline]

Pollock, V. P., Mcgettigan, J., Cabrero, P., Maudlin, I. M., Dow, J. A. T. and Davies, S. A. (2004). Conservation of capa peptide-induced nitric oxide signalling in Diptera. J. Exp. Biol. 207,4135 -4145.[Abstract/Free Full Text]

Predel, R. and Wegener, C. (2006). Biology of the CAPA peptides in insects. Cell. Mol. Life Sci. 63,2477 -2490.[CrossRef][Medline]

Quinlan, M. C. and O'Donnell, M. J. (1998). Anti-diuresis in the blood-feeding insect Rhodnius prolixus Stal: antagonistic actions of cAMP and cGMP and the role of organic acid transport. J. Insect Physiol. 44,561 -568.[CrossRef][Medline]

Radford, J. C., Davies, S. A. and Dow, J. A. T. (2002). Systematic GPCR analysis in Drosophila melanogaster identifies a leucokinin receptor with novel roles. J. Biol. Chem. 277,38810 -38817.[Abstract/Free Full Text]

Radford, J. C., Terhzaz, S., Cabrero, P., Davies, S. A. and Dow, J. A. T. (2004). Functional characterisation of the Anopheles leucokinins and their cognate G-protein coupled receptor. J. Exp. Biol. 207,4573 -4586.[Abstract/Free Full Text]

Rong, Y. S. and Golic, K. G. (2000). Gene targeting by homologous recombination in Drosophila.Science 288,2013 -2018.[Abstract/Free Full Text]

Rong, Y. S., Titen, S. W., Xie, H. B., Golic, M. M., Bastiani, M., Bandyopadhyay, P., Olivera, B. M., Brodsky, M., Rubin, G. M. and Golic, K. G. (2002). Targeted mutagenesis by homologous recombination in D. melanogaster. Genes Dev. 16,1568 -1581.[Abstract/Free Full Text]

Rosay, P., Davies, S. A., Yu, Y., Sozen, M. A., Kaiser, K. and Dow, J. A. T. (1997). Cell-type specific calcium signalling in a Drosophila epithelium. J. Cell Sci. 110,1683 -1692.[Abstract]

Salkoff, L., Baker, K., Butler, A., Covarrubius, M., Pak, M. D. and Wei, A. (1992). An essential "set" of K⁺ channels conserved in flies, mice and humans. Trends Neurosci. 15,161 -166.[CrossRef][Medline]

Sentry, J. W., Goodwin, S. F., Milligan, C. D., Duncanson, A., Yang, M. and Kaiser, K. (1994). Reverse genetics of Drosophila brain structure and function. Prog. Neurobiol. 42,299 -308.[CrossRef][Medline]

Sözen, M. A., Armstrong, J. D., Yang, M. Y., Kaiser, K. and Dow, J. A. T. (1997). Functional domains are specified to single-cell resolution in a Drosophila epithelium. Proc. Natl. Acad. Sci. USA 94,5207 -5212.[Abstract/Free Full Text]

Strange, K. (2003). From genes to integrative physiology: ion channel and transporter biology in Caenorhabditis elegans.Physiol. Rev. 83,377 -415.[Abstract/Free Full Text]

Strange, K. (2007). Revisiting the Krogh Principle in the post-genome era: Caenorhabditis elegans as a model system for integrative physiology research. J. Exp. Biol. 210,1622 -1631.[Abstract/Free Full Text]

Suster, M. L., Seugnet, L., Bate, M. and Sokolowski, M. B. (2004). Refining GAL4-driven transgene expression in Drosophila with a GAL80 enhancertrap. Genesis 39,240 -245.[CrossRef][Medline]

Terhzaz, S., O'Connell, F. C., Pollock, V. P., Kean, L., Davies, S. A., Veenstra, J. A. and Dow, J. A. T. (1999). Isolation and characterization of a leucokinin-like peptide of Drosophila melanogaster. J. Exp. Biol. 202,3667 -3676.[Abstract]

Terhzaz, S., Southall, T. D., Lilley, K. S., Kean, L., Allan, A. K., Davies, S. A. and Dow, J. A. T. (2006). Differential gel electrophoresis and transgenic mitochondrial calcium reporters demonstrate spatiotemporal filtering in calcium control of mitochondria. J. Biol. Chem. 281,18849 -18858.[Abstract/Free Full Text]

Wang, J. and Barr, M. M. (2005). RNA interference in Caenorhabditis elegans. Meth. Enzymol. 392, 36-55.[Medline]

Wang, J., Kean, L., Yang, J., Allan, A. K., Davies, S. A., Herzyk, P. and Dow, J. A. T. (2004). Function-informed transcriptome analysis of Drosophila renal tubule. Genome Biol. 5,R69 .[CrossRef][Medline]

Weltens, R., Leyssens, A., Zhang, A. L., Lohhrmann, E., Steels, P. and van Kerkhove, E. (1992). Unmasking of the apical electrogenic H pump in isolated Malpighian tubules (Formica polyctena) by the use of barium. Cell. Physiol. Biochem. 2,101 -116.

Wiehart, U. I., Klein, G., Steels, P., Nicolson, S. W. and Van Kerkhove, E. (2003a). K⁺ transport in Malpighian tubules of Tenebrio molitor L.: is a K_ATP channel involved? J. Exp. Biol. 206,959 -965.[Abstract/Free Full Text]

Wiehart, U. I., Nicolson, S. W. and Van Kerkhove, E. (2003b). The effects of endogenous diuretic and antidiuretic peptides and their second messengers in the Malpighian tubules of Tenebrio molitor: an electrophysiological study. J. Insect Physiol. 49,955 -965.[CrossRef][Medline]

Xu, T. and Harrison, S. D. (1994). Mosaic analysis using FLP recombinase. Methods Cell Biol. 44,655 -681.[Medline]

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