Identification and functional analysis of antifungal immune response genes in Drosophila

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-kappaB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.     

Characterizing dopaminergic neuron vulnerability using genome-wide analysis

Parkinson’s disease (PD) is primarily characterized by the loss of dopaminergic (DA) neurons in the brain. However, little is known about why DA neurons are selectively vulnerable to PD. To identify genes that are associated with DA neuron loss, we screened through 201 wild-caught populations of Drosophila melanogaster as part of the Drosophila Genetic Reference Panel. Here, we identify the top-associated genes containing single-nucleotide polymorphisms that render DA neurons vulnerable. These genes were further analyzed by using mutant analysis and tissue-specific knockdown for functional validation. We found that this loss of DA neurons caused progressive locomotor dysfunction in mutants and gene knockdown analysis. The identification of genes associated with the progressive loss of DA neurons should help to uncover factors that render these neurons vulnerable in PD, and possibly develop strategies to make these neurons more resilient.     

Fruit flies and intellectual disability

Mental retardation - more commonly known nowadays as intellectual disability - is a severe neurological condition affecting 3% of the general population. As a result of analysis of familial cases and recent advances in clinical genetic testing great strides have been made in our understanding of the genetic etiologies of mental retardation. Nonetheless, no treatment is currently clinically available to patients suffering from intellectual disability. Several animal models have been used in the study of memory and cognition. Established paradigms in Drosophila have recently captured cognitive defects in fly mutants for orthologs of genes involved in human intellectual disability. We review here three protocols designed to understand the molecular genetic basis of learning and memory in Drosophila and the genes identified so far with relation to mental retardation. In addition, we explore the mental retardation genes for which evidence of neuronal dysfunction other than memory has been established in Drosophila. Finally, we summarize the findings in Drosophila for mental retardation genes for which no neuronal information is yet available. All in all, this review illustrates the impressive overlap between genes identified in human mental retardation and genes involved in physiological learning and memory.     

A large-scale screen for mutagen-sensitive loci in Drosophila

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212-mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314-mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215(ZIII-2059), that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.     

The Genetics of CAENORHABDITIS ELEGANS

Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.     

In silico identification of new secretory peptide genes in Drosophila melanogaster

Bioactive peptides play critical roles in regulating most biological processes in animals. The elucidation of the amino acid sequence of these regulatory peptides is crucial for our understanding of animal physiology. Most of the (neuro)peptides currently known were identified by purification and subsequent amino acid sequencing. With the entire genome sequence of some animals now available, it has become possible to predict novel putative peptides. In this way, BLAST (Basic Local Alignment Searching Tool) analysis of the Drosophila melanogaster genome has allowed annotation of 36 secretory peptide genes so far. Peptide precursor genes are, however, poorly predicted by this algorithm, thus prompting an alternative approach described here. With the described searching program we scanned the Drosophila genome for predicted proteins with the structural hallmarks of neuropeptide precursors. As a result, 76 additional putative secretory peptide genes were predicted in addition to the 43 annotated ones. These putative (neuro)peptide genes contain conserved motifs reminiscent of known neuropeptides from other animal species. Peptides that display sequence similarities to the mammalian vasopressin, atrial natriuretic peptide, and prolactin precursors and the invertebrate peptides orcokinin, prothoracicotropic hormones, trypsin modulating oostatic factor, and Drosophila immune induced peptides (DIMs) among others were discovered. Our data hence provide further evidence that many neuropeptide genes were already present in the ancestor of Protostomia and Deuterostomia prior to their divergence. This bioinformatic study opens perspectives for the genome-wide analysis of peptide genes in other eukaryotic model organisms.     

Genetic dissection of meiotic cytokinesis in Drosophila males

We have used Drosophila male meiosis as a model system for genetic dissection of the cytokinesis mechanism. Drosophila mutants defective in meiotic cytokinesis can be easily identified by their multinucleate spermatids. Moreover, the large size of meiotic spindles allows characterization of mutant phenotypes with exquisite cytological resolution. We have screened a collection of 1955 homozygous mutant male sterile lines for those with multinucleate spermatids, and thereby identified mutations in 19 genes required for cytokinesis. These include 16 novel loci and three genes, diaphanous, four wheel drive, and pebble, already known to be involved in Drosophila cytokinesis. To define the primary defects leading to failure of cytokinesis, we analyzed meiotic divisions in male mutants for each of these 19 genes. Examination of preparations stained for tubulin, anillin, KLP3A, and F-actin revealed discrete defects in the components of the cytokinetic apparatus, suggesting that these genes act at four major points in a stepwise pathway for cytokinesis. Our results also indicated that the central spindle and the contractile ring are interdependent structures that interact throughout cytokinesis. Moreover, our genetic and cytological analyses provide further evidence for a cell type-specific control of Drosophila cytokinesis, suggesting that several genes required for meiotic cytokinesis in males are not required for mitotic cytokinesis.     

DNA repair defects and other (mus)takes in Drosophila melanogaster.

Preservation of the structural integrity of DNA in any organism is crucial to its health and survival. Such preservation is achieved by an extraordinary cellular arsenal of damage surveillance and repair functions, many of which are now being defined at the gene and protein levels. Mutants hypersensitive to the killing effects of DNA-damaging agents have been instrumental in helping to identify DNA repair-related genes and to elucidate repair mechanisms. In Drosophila melanogaster, such strains are generally referred to as mutagen-sensitive (mus) mutants and currently define more than 30 genetic loci. Whereas most mus mutants have been recovered on the basis of hypersensitivity to the monofunctional alkylating agent methyl methanesulfonate, they nevertheless constitute a phenotypically diverse group, with many mutants having effects beyond mutagen sensitivity. These phenotypes include meiotic dysfunctions, somatic chromosome instabilities, chromatin abnormalities, and cell proliferation defects. Within the last few years numerous mus and other DNA repair-related genes of Drosophila have been molecularly cloned, providing new insights into the functions of these genes. This article outlines strategies for isolating mus mutations and reviews recent advances in the Drosophila DNA repair field, emphasizing mutant analysis and gene cloning.     

Genes that fight infection: what the Drosophila genome says about animal immunity

From deciphering the principles of heredity to identifying the genes that control development, the fruit fly Drosophila melanogaster is being used to deconstruct an increasing number of biological processes. Genetic studies of Drosophila responses to microbial infection have identified regulators of innate immunity that are functionally conserved in mammals. These recent findings highlight the ancient origins of animal immune responses and demonstrate the potential of Drosophila for dissecting host-pathogen interactions. The sequencing of the Drosophila genome both enhances genetic approaches and provides new clues for the identification of key components of innate immunity. This article summarizes how information gained from genomic analysis contributes to our understanding of how animals cope with infectious disease.     

Drosophila homologs of FANCD2 and FANCL function in DNA repair

Fanconi anemia (FA) is a genetically heterogeneous disease characterized by developmental defects, progressive bone marrow failure and cancer susceptibility. Cells derived from patients with FA show spontaneous chromosomal aberrations and hypersensitivity to cross-linking agents, indicating a cellular defect in DNA repair. Among the 12 FA genes, only FANCD2, FANCL and FANCM have Drosophila homologs. Given this difference between the human and Drosophila FA pathways, it is unknown whether the fly homologs function in DNA repair. Here, we report that knockdown of Drosophila FANCD2 or FANCL leads to specific hypersensitivity to cross-linking agents. Further analysis revealed that FANCD2 and FANCL function in a linear pathway with FANCL being necessary for the monoubiquitination of FANCD2. FANCD2 mutants also exhibited the same defect in the ionizing radiation-inducible S-phase checkpoint that is seen in mammalian cells deficient for this gene. Finally, in an assay for inactivating mutations, FANCD2 mutants have an elevated mutation rate in response to nitrogen mustard, indicating that these flies are hypermutable. Taken together, these data demonstrate that Drosophila FANCD2 and FANCL play a critical role in DNA repair. Because of the lack of other FA genes, further studies will determine whether the conserved FA genes function as the minimal machinery or whether additional genes are involved in the Drosophila FA pathway.