Julian A. T. Dow

Using FlyAtlas to identify better Drosophila melanogaster models of human disease

FlyAtlas, a new online resource, provides the most comprehensive view yet of expression in multiple tissues of Drosophila melanogaster. Meta-analysis of the data shows that a significant fraction of the genome is expressed with great tissue specificity in the adult, demonstrating the need for the functional genomic community to embrace a wide range of functional phenotypes. Well-known developmental genes are often reused in surprising tissues in the adult, suggesting new functions. The homologs of many human genetic disease loci show selective expression in the Drosophila tissues analogous to the affected human tissues, providing a useful filter for potential candidate genes. Additionally, the contributions of each tissue to the whole-fly array signal can be calculated, demonstrating the limitations of whole-organism approaches to functional genomics and allowing modeling of a simple tissue fractionation procedure that should improve detection of weak or tissue-specific signals.

Insect Midgut Function

Publisher Summary This chapter presents recent progress in the study of the midgut, particularly with reference to ion and water transport. However, the midgut does not function in isolation, and so the attempt is to fit the role of the midgut into the context of the operation of the gut as a whole. The chapter considers the cockroach as an example of a classic generalist and looks at scavenging dipteran larvae, mainly aquatic. Midgut function is described in various types of insects such as solid/plant feeders (phytophagous insects), solid/animal feeders (carnivores), liquid/animal feeders (bloodsuckers), and liquid/plant feeders (sap and nectar feeders). The chapter also discusses the process of digestion of cellulose in insects. There are four categories of evidence that an insect can digest cellulose. It could survive on a diet of pure synthetic cellulose; it could be shown to incorporate and metabolize label from radioactive cellulose in the food; it could be shown to break down synthetic crystalline cellulose; or it could be shown that there is less cellulose in the excreta than was consumed in the food.

Function-informed transcriptome analysis of Drosophila renal tubule

BackgroundComprehensive, tissue-specific, microarray analysis is a potent tool for the identification of tightly defined expression patterns that might be missed in whole-organism scans. We applied such an analysis to Drosophila melanogaster Malpighian (renal) tubule, a defined differentiated tissue.ResultsThe transcriptome of the D. melanogaster Malpighian tubule is highly reproducible and significantly different from that obtained from whole-organism arrays. More than 200 genes are more than 10-fold enriched and over 1,000 are significantly enriched. Of the top 200 genes, only 18 have previously been named, and only 45% have even estimates of function. In addition, 30 transcription factors, not previously implicated in tubule development, are shown to be enriched in adult tubule, and their expression patterns respect precisely the domains and cell types previously identified by enhancer trapping. Of Drosophila genes with close human disease homologs, 50 are enriched threefold or more, and eight enriched 10-fold or more, in tubule. Intriguingly, several of these diseases have human renal phenotypes, implying close conservation of renal function across 400 million years of divergent evolution.ConclusionsFrom those genes that are identifiable, a radically new view of the function of the tubule, emphasizing solute transport rather than fluid secretion, can be obtained. The results illustrate the phenotype gap: historically, the effort expended on a model organism has tended to concentrate on a relatively small set of processes, rather than on the spread of genes in the genome.

The Developmental, Molecular, and Transport Biology of Malpighian Tubules

Molecular biology is reaching new depths in our understanding of the development and physiology of Malpighian tubules. In Diptera, Malpighian tubules derive from ectodermal cells that evaginate from the primitive hindgut and subsequently undergo a sequence of orderly events that culminates in an active excretory organ by the time the larva takes its first meal. Thereafter, the tubules enlarge by cell growth. Just as modern experimental strategies have illuminated the development of tubules, genomic, transcriptomic, and proteomic studies have uncovered new tubule functions that serve immune defenses and the breakdown and renal clearance of toxic substances. Moreover, genes associated with specific diseases in humans are also found in flies, some of which, astonishingly, express similar pathophenotypes. However, classical experimental approaches continue to show their worth by distinguishing between -omic possibilities and physiological reality while providing further detail about the rapid regulation of the transport pathway through septate junctions and the reversible assembly of proton pumps.

An Extracellular microelectrode Array for monitoring electrogenic cells in culture

This paper describes a planar array of microelectrodes developed for monitoring the electrical activity of cells in culture. The device allows the incorporation of surface topographical features in an insulating layer above the electrodes. Semiconductor technology is employed for the fabrication of the gold electrodes and for the deposition and patterning of an insulating layer of silicon nitride. The electrodes have been tested using a cardiac cell culture of chick embryo myocytes, and the physical beating of the cultured cells correlated with the simultaneous extracellular voltage measurements obtained. It was found that extracellular stimulation of the cells was possible via the same electrodes used for recording.

A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling.

SUMMARY The Drosophila orphan G protein-coupled receptor encoded by CG17415 is related to members of the calcitonin receptor-like receptor (CLR) family. In mammals, signaling from CLR receptors depend on accessory proteins, namely the receptor activity modifying proteins (RAMPs) and receptor component protein (RCP). We tested the possibility that this Drosophila CLR might also require accessory proteins for proper function and we report that co-expression of the mammalian or Drosophila RCP or mammalian RAMPs permitted neuropeptide diuretic hormone 31 (DH31) signaling from the CG17415 receptor. RAMP subtype expression did not alter the pharmacological profile of CG17415 activation. CG17415 antibodies revealed expression within the principal cells of Malpighian tubules, further implicating DH31 as a ligand for this receptor. Immunostaining in the brain revealed an unexpected convergence of two distinct DH signaling pathways. In both the larval and adult brain, most DH31 receptor-expressing neurons produce the neuropeptide corazonin, and also express the CRFR-related receptor CG8422, which is a receptor for the neuropeptide diuretic hormone 44 (DH44). There is extensive convergence of CRF and CGRP signaling within vertebrates and we report a striking parallel in Drosophila involving DH44 (CRF) and DH31 (CGRP). Therefore, it appears that both the molecular details as well as the functional organization of CGRP signaling have been conserved.

FlyAtlas: database of gene expression in the tissues of Drosophila melanogaster

The FlyAtlas resource contains data on the expression of the genes of Drosophila melanogaster in different tissues (currently 25—17 adult and 8 larval) obtained by hybridization of messenger RNA to Affymetrix Drosophila Genome 2 microarrays. The microarray probe sets cover 13 250 Drosophila genes, detecting 12 533 in an unambiguous manner. The data underlying the original web application (http://flyatlas.org) have been restructured into a relational database and a Java servlet written to provide a new web interface, FlyAtlas 2 (http://flyatlas.gla.ac.uk/), which allows several additional queries. Users can retrieve data for individual genes or for groups of genes belonging to the same or related ontological categories. Assistance in selecting valid search terms is provided by an Ajax ‘autosuggest’ facility that polls the database as the user types. Searches can also focus on particular tissues, and data can be retrieved for the most highly expressed genes, for genes of a particular category with above-average expression or for genes with the greatest difference in expression between the larval and adult stages. A novel facility allows the database to be queried with a specific gene to find other genes with a similar pattern of expression across the different tissues.

Analysis and inactivation of vha55, the gene encoding the vacuolar ATPase B-subunit in Drosophila melanogaster reveals a larval lethal phenotype.

Vacuolar ATPases play major roles in endomembrane and plasma membrane proton transport in eukaryotes. A Drosophila melanogaster cDNA encoding vha55, the 55-kDa vacuolar ATPase (V-ATPase) regulatory B-subunit, was characterized and mapped to 87C2-4 on chromosome 3R. A fly line was identified that carried a single lethal P-element insertion within the coding portion of gene, and its LacZ reporter gene revealed elevated expression in Malpighian tubules, rectum, antennal palps, and oviduct, regions where V-ATPases are believed to play a plasma membrane, rather than an endomembrane, role. The P-element vha55 insertion was shown to be allelic to a known lethal complementation group l(3)SzA (= l(3)87Ca) at 87C, for which many alleles have been described previously. Deletions of the locus have been shown to be larval lethal, whereas point mutations show a range of phenotypes from subvital to embryonic lethal, implying that severe alleles confer a partial dominant negative phenotype. The P-element null allele of vha55 was shown also to suppress ectopic sex combs in Polycomb males, suggesting that transcriptional silencing may be modulated by genes other than those with known homeotic or DNA binding functions.

Insights into the Malpighian tubule from functional genomics

SUMMARY Classical physiological study of the Malpighian tubule has led to a detailed understanding of fluid transport and its control across several species. With the sequencing of the Drosophila genome, and the concurrent development of post-genomic technologies such as microarrays, proteomics, metabolomics and systems biology, completely unexpected roles for the insect Malpighian tubule have emerged. As the insect body plan is simpler than that of mammals, tasks analogous to those performed by multiple mammalian organ systems must be shared out among insect tissues. As well as the classical roles in osmoregulation, the Malpighian tubule is highly specialized for organic solute transport, and for metabolism and detoxification. In Drosophila, the adult Malpighian tubule is the key tissue for defence against insecticides such as DDT; and it can also detect and mount an autonomous defence against bacterial invasion. While it is vital to continue to set insights obtained in Drosophila into the context of work in other species, the combination of post-genomic technologies and physiological validation can provide insights that might not otherwise have been apparent for many years.

Drosophila provides rapid modeling of renal development, function, and disease

The evolution of specialized excretory cells is a cornerstone of the metazoan radiation, and the basic tasks performed by Drosophila and human renal systems are similar. The development of the Drosophila renal (Malpighian) tubule is a classic example of branched tubular morphogenesis, allowing study of mesenchymal-to-epithelial transitions, stem cell-mediated regeneration, and the evolution of a glomerular kidney. Tubule function employs conserved transport proteins, such as the Na(+), K(+)-ATPase and V-ATPase, aquaporins, inward rectifier K(+) channels, and organic solute transporters, regulated by cAMP, cGMP, nitric oxide, and calcium. In addition to generation and selective reabsorption of primary urine, the tubule plays roles in metabolism and excretion of xenobiotics, and in innate immunity. The gene expression resource FlyAtlas.org shows that the tubule is an ideal tissue for the modeling of renal diseases, such as nephrolithiasis and Bartter syndrome, or for inborn errors of metabolism. Studies are assisted by uniquely powerful genetic and transgenic resources, the widespread availability of mutant stocks, and low-cost, rapid deployment of new transgenics to allow manipulation of renal function in an organotypic context.