Kevin M.C. O'Dell

Functional dissection of the drosophila mushroom bodies by selective feminization ofagenetically defined subcompartments

Relatively little is known about the neural circuitry underlying sex-specific behaviors. We have expressed the feminizing gene transformer in genetically defined subregions of the brain of male Drosophila, and in particular within different domains of the mushroom bodies. Mushroom bodies are phylogenetically conserved insect brain centers implicated in associative learning and various other aspects of behavior. Expression of transformer in lines that mark certain subsets of mushroom body intrinsic neurons, and in a line that marks a component of the antennal lobe, causes males to exhibit nondiscriminatory sexual behavior: they court mature males in addition to females. Expression of transformer in other mushroom body domains, and in control lines, has no such effect. Our data support the view that genetically defined subsets of mushroom body intrinsic neurons perform different functional roles.

Expression of the Ciona intestinalis Alternative Oxidase (AOX) in Drosophila Complements Defects in Mitochondrial Oxidative Phosphorylation

Defects in mitochondrial OXPHOS are associated with diverse and mostly intractable human disorders. The single-subunit alternative oxidase (AOX) found in many eukaryotes, but not in arthropods or vertebrates, offers a potential bypass of the OXPHOS cytochrome chain under conditions of pathological OXPHOS inhibition. We have engineered Ciona intestinalis AOX for conditional expression in Drosophila melanogaster. Ubiquitous AOX expression produced no detrimental phenotype in wild-type flies. However, mitochondrial suspensions from AOX-expressing flies exhibited a significant cyanide-resistant substrate oxidation, and the flies were partially resistant to both cyanide and antimycin. AOX expression was able to complement the semilethality of partial knockdown of both cyclope (COXVIc) and the complex IV assembly factor Surf1. It also rescued the locomotor defect and excess mitochondrial ROS production of flies mutated in dj-1beta, a Drosophila homolog of the human Parkinsons disease gene DJ1. AOX appears to offer promise as a wide-spectrum therapeutic tool in OXPHOS disorders.

Gene Expression in a Drosophila Model of Mitochondrial Disease

Background A point mutation in the Drosophila gene technical knockout (tko), encoding mitoribosomal protein S12, was previously shown to cause a phenotype of respiratory chain deficiency, developmental delay, and neurological abnormalities similar to those presented in many human mitochondrial disorders, as well as defective courtship behavior. Methodology/Principal Findings Here, we describe a transcriptome-wide analysis of gene expression in tko25t mutant flies that revealed systematic and compensatory changes in the expression of genes connected with metabolism, including up-regulation of lactate dehydrogenase and of many genes involved in the catabolism of fats and proteins, and various anaplerotic pathways. Gut-specific enzymes involved in the primary mobilization of dietary fats and proteins, as well as a number of transport functions, were also strongly up-regulated, consistent with the idea that oxidative phosphorylation OXPHOS dysfunction is perceived physiologically as a starvation for particular biomolecules. In addition, many stress-response genes were induced. Other changes may reflect a signature of developmental delay, notably a down-regulation of genes connected with reproduction, including gametogenesis, as well as courtship behavior in males; logically this represents a programmed response to a mitochondrially generated starvation signal. The underlying signalling pathway, if conserved, could influence many physiological processes in response to nutritional stress, although any such pathway involved remains unidentified. Conclusions/Significance These studies indicate that general and organ-specific metabolism is transformed in response to mitochondrial dysfunction, including digestive and absorptive functions, and give important clues as to how novel therapeutic strategies for mitochondrial disorders might be developed.

Metazoan nuclear genes for mitoribosomal protein S12

We have characterized nuclear genes for mitoribosomal protein S12 (mt-rps12) a major component of the ribosomal accuracy centre, in human, mouse and Drosophila melanogaster. In human and Drosophila, and probably also in mouse, there is a single intron within the coding region, located in the mitochondrial targeting pre-sequence. In humans, the mRNA structure is highly suggestive of translational regulation. In all three species, there is an amino-acid substitution with respect to eubacterial homologues in a residue implicated in aminoglycoside resistance. The only viable mutant allele of the Drosophila gene, associated with a bang-sensitive phenotype (paralysis upon mechanical vibration, arising from a mechanoreceptor cell defect) also has a novel substitution in a conserved region implicated in translational fidelity. Given the involvement of the mitoribosomal accuracy centre in human sensorineural deafness by virtue of rRNA mutations, our results indicate that this fly mutant may be a useful animal model of this disorder, and earmark the gene for mt-rps12 as a candidate in human hearing impairment.

The Dominant Cold-Sensitive Out-Cold Mutants of Drosophila melanogaster Have Novel Missense Mutations in the Voltage-Gated Sodium Channel Gene paralytic

Here we report the molecular characterization of Out-cold (Ocd) mutants of Drosophila melanogaster, which produce a dominant, X-linked, cold-sensitive paralytic phenotype. From its initial 1.5-Mb cytological location within 13F1-16A2, P-element and SNP mapping reduced the Ocd critical region to <100 kb and to six candidate genes: hangover, CG9947, CG4420, eIF2a, Rbp2, and paralytic (para). Complementation testing with para null mutations strongly suggests Ocd and para are allelic, as does gene rescue of Ocd semilethality with a wild-type para transgene. Pesticide resistance and electrophysiological phenotypes of Ocd mutants support this conclusion. The para gene encodes a voltage-gated sodium channel. Sequencing the Ocd lines revealed mutations within highly conserved regions of the para coding sequence, in the transmembrane segment S6 of domain III (I1545M and T1551I), and in the linker between domains III and IV (G1571R), the location of the channel inactivation gate. The G1571R mutation is of particular interest as mutations of the orthologous residue (G1306) in the human skeletal muscle sodium channel gene SCN4A are associated with cases of periodic paralysis and myotonia, including the human cold-sensitive disorder paramyotonia congenita. The mechanisms by which sodium channel mutations cause cold sensitivity are not well understood. Therefore, in the absence of suitable vertebrate models, Ocd provides a system in which genetic, molecular, physiological, and behavioral tools can be exploited to determine mechanisms underlying sodium channel periodic paralyses.

Female receptivity phenotype of icebox mutants caused by a mutation in the L1-type cell adhesion molecule neuroglian.

Relatively little is known about the genes and brain structures that enable virgin female Drosophila to make the decision to mate or not. Classical genetic approaches have identified several mutant females that have a reluctance‐to‐mate phenotype, but most of these have additional behavioral defects. However, the icebox (ibx) mutation was previously reported to lower the sexual receptivity of females, without apparently affecting any other aspect of female behavior. We have shown that the ibx mutation maps to the 7F region of the Drosophila X chromosome to form a complex complementation group with both lethal and viable alleles of neuroglian (nrg). The L1‐type cell adhesion molecule encoded by nrg consists of six immunoglobulin‐like domains, five fibronectin‐like domains, one transmembrane domain and one alternatively spliced intracellular domain. The ibx strain has a missense mutation causing a glycine‐to‐arginine change at amino acid 92 in the first immunoglobulin domain of nrg. Defects in the central brain of ibx mutants are similar to those observed in another nrg mutant, central brain deranged1 (ceb1). However, both ceb1 homozygous and ceb1/ibx heterozygous females are receptive. The expression of a transgene containing the non‐neural isoform of nrg rescues both the receptivity and the brain structure phenotypes of ibx females.

Gene dosage and selective expression modify phenotype in a Drosophila model of human mitochondrial disease.

Human mitochondrial disease manifests with a wide range of clinical phenotypes of varying severity. To create a model for these disorders, we have manipulated the Drosophila gene technical knockout, encoding mitoribosomal protein S12. Various permutations of endogenous and transgenic alleles create a range of phenotypes, varying from larval developmental arrest through to mild neurological defects in the adult, and also mimic threshold effects associated with human mtDNA disease. Nuclear genetic background influences mutant phenotype by a compensatory mechanism affecting mitochondrial RNA levels. Selective expression of the wild-type allele indicates critical times and cell-types in development, in which mitochondrial protein synthesis deficiency leads to specific phenotypic outcomes.

The Effects of Ectopic White and Transformer Expression on Drosophila Courtship Behavior

The sex determining genes of Drosophila males and females function to establish the potential for sex-specific behaviors. Previous studies suggest that ectopic GAL4-directed misexpression of the female-specific isoform of the sex-determining gene transformer (tra) in specific sub-domains of an otherwise male brain can lead to bisexual courtship behavior, thus identifying brain domains that may mediate sex-specific behavior. However, expression of mini-white, the marker gene used in both P{GAL4} and P{UASG} constructs, also induces males to court other males, questioning whether GAL4-mediated tra expression alone can induce bisexual behavior. Here we demonstrate the consequences of inducing mutations in the mini-white genes within P{GAL4} and P{UASG} constructs to generate flies in which a white mutant phenotype is revealed. In these mini-white mutant strains, P{GAL4}-mediated transformer expression alone is both sufficient and necessary to generate bisexual behavior. In addition, using RT-PCR, we reveal the presence of female transcripts of doublesex and fruitless in the brains of otherwise male (XY) flies exhibiting P{GAL4}-directed tra-expression, demonstrating that P{GAL4}-directed tra is functional at the molecular level. We conclude that P{GAL4}-directed misexpression of tra is responsible for the bisexual behavior previously described and that this is mediated via sex-specific splicing of dsx and fru. Our results support the validity of such strategies for identifying regions of the fly brain that underlie sex-specific behaviors.

Phenotypic rescue of a Drosophila model of mitochondrial ANT1 disease

A point mutation in the Drosophila gene that codes for the major adult isoform of adenine nuclear translocase (ANT) represents a model for human diseases that are associated with ANT insufficiency [stress-sensitive B1 (sesB1)]. We characterized the organismal, bioenergetic and molecular phenotype of sesB1 flies then tested strategies to compensate the mutant phenotype. In addition to developmental delay and mechanical-stress-induced seizures, sesB1 flies have an impaired response to sound, defective male courtship, female sterility and curtailed lifespan. These phenotypes, excluding the latter two, are shared with the mitoribosomal protein S12 mutant, tko25t. Mitochondria from sesB1 adults showed a decreased respiratory control ratio and downregulation of cytochrome oxidase. sesB1 adults exhibited ATP depletion, lactate accumulation and changes in gene expression that were consistent with a metabolic shift towards glycolysis, characterized by activation of lactate dehydrogenase and anaplerotic pathways. Females also showed downregulation of many genes that are required for oogenesis, and their eggs, although fertilized, failed to develop to the larval stages. The sesB1 phenotypes of developmental delay and mechanical-stress-induced seizures were alleviated by an altered mitochondrial DNA background. Female sterility was substantially rescued by somatic expression of alternative oxidase (AOX) from the sea squirt Ciona intestinalis, whereas AOX did not alleviate developmental delay. Our findings illustrate the potential of different therapeutic strategies for ANT-linked diseases, based on alleviating metabolic stress.

Abnormal courtship conditioning in males mutant for the RI regulatory subunit of Drosophila protein kinase A.

The previously described site-selected P-element mutagenesis of a Drosophila gene encoding the regulatory subunit of cAMP-dependent protein kinase generates mutants that have defective behavior in the olfactory learning test. Here we describe the effect of the same mutations in a courtship conditioning assay. Wild-type males can distinguish between virgin females (which they court vigorously), and fertilized females (which they court less vigorously). After exposure to fertilized females, wild-type males modify their behavior by decreasing courtship to subsequent target virgins, an effect that may last for many hours. Like wild-type males, PKA-RI mutant males are also able to distinguish between virgin and fertilized females. PKA-RI males also modify their behavior towards virgin females after prior exposure to a fertilized female, but such an effect is short-lived, suggesting a defect in memory rather than learning. We also show that under these conditions the behavior of PKA-RI males is similar to that of amnesiac, dunce and rutabaga males.