Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.     

Homology-based annotation yields 1,042 new candidate genes in the Drosophila melanogaster genome

The approach to annotating a genome critically affects the number and accuracy of genes identified in the genome sequence. Genome annotation based on stringent gene identification is prone to underestimate the complement of genes encoded in a genome. In contrast, over-prediction of putative genes followed by exhaustive computational sequence, motif and structural homology search will find rarely expressed, possibly unique, new genes at the risk of including non-functional genes. We developed a two-stage approach that combines the merits of stringent genome annotation with the benefits of over-prediction. First we identify plausible genes regardless of matches with EST, cDNA or protein sequences from the organism (stage 1). In the second stage, proteins predicted from the plausible genes are compared at the protein level with EST, cDNA and protein sequences, and protein structures from other organisms (stage 2). Remote but biologically meaningful protein sequence or structure homologies provide supporting evidence for genuine genes. The method, applied to the Drosophila melanogaster genome, validated 1,042 novel candidate genes after filtering 19,410 plausible genes, of which 12,124 matched the original 13,601 annotated genes. This annotation strategy is applicable to genomes of all organisms, including human.     

Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint

In both yeast and mammals, uncapped telomeres activate the DNA damage response (DDR) and undergo end-to-end fusion. Previous work has shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions. Here we show that HOAP-depleted telomeres activate both the DDR and the spindle assembly checkpoint (SAC). The cell cycle arrest elicited by the DDR was alleviated by mutations in mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50 but not by mutations in tefu (ATM). The SAC was partially overridden by mutations in zw10 (also known as mit(1)15) and bubR1, and also by mutations in mei-41, mus304, rad50, grp and tefu. As expected from SAC activation, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulated at the kinetochores of cav mutant cells. Notably, BubR1 also accumulated at cav mutant telomeres in a mei-41-, mus304-, rad50-, grp- and tefu-dependent manner. Our results collectively suggest that recruitment of BubR1 by dysfunctional telomeres inhibits Cdc20-APC function, preventing the metaphase-to-anaphase transition.     

Genomic and functional evolution of the Drosophila melanogaster sperm proteome

In addition to delivering a haploid genome to the egg, sperm have additional critical functions, including egg activation, origination of the zygote centrosome and delivery of paternal factors. Despite this, existing knowledge of the molecular basis of sperm form and function is limited. We used whole-sperm mass spectrometry to identify 381 proteins of the Drosophila melanogaster sperm proteome (DmSP). This approach identified mitochondrial, metabolic and cytoskeletal proteins, in addition to several new functional categories. We also observed nonrandom genomic clustering of sperm genes and underrepresentation on the X chromosome. Identification of widespread functional constraint on the proteome indicates that sexual selection has had a limited role in the overall evolution of D. melanogaster sperm. The relevance of the DmSP to the study of mammalian sperm function and fertilization mechanisms is demonstrated by the identification of substantial homology between the DmSP and proteins of the mouse axoneme accessory structure.     

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.     

Probing long-distance regulatory interactions in the Drosophila melanogaster bithorax complex using Dam identification

A cis-regulatory region of nearly 300 kb controls the expression of the three bithorax complex (BX-C) homeotic genes: Ubx, abd-A and Abd-B. Interspersed between the numerous enhancers and silencers within the complex are elements called domain boundaries. Recently, many pieces of evidence have suggested that boundaries function to create autonomous domains by interacting among themselves and forming chromatin loops. In order to test this hypothesis, we used Dam identification to probe for interactions between the Fab-7 boundary and other regions in the BX-C. We were surprised to find that the targeting of Dam methyltransferase (Dam) to the Fab-7 boundary results in a strong methylation signal at the Abd-Bm promoter, approximately 35 kb away. Moreover, this methylation pattern is found primarily in the tissues where Abd-B is not expressed and requires an intact Fab-7 boundary. Overall, our work provides the first documented example of a dynamic, long-distance physical interaction between distal regulatory elements within a living, multicellular organism.     

Pleiotropic fitness effects of the Tre1-Gr5a region in Drosophila melanogaster

The abundance of transposable elements and DNA repeat sequences in mammalian genomes raises the question of whether such insertions represent passive evolutionary baggage or may influence the expression of complex traits. We addressed this question in Drosophila melanogaster, in which the effects of single transposable elements on complex traits can be assessed in genetically identical individuals reared in controlled environments. Here we demonstrate that single P-element insertions in the intergenic region between the gustatory receptor 5a (Gr5a, also known as Tre) and trapped in endoderm 1 (Tre1), which encodes an orphan receptor, exert complex pleiotropic effects on fitness traits, including selective nutrient intake, life span, and resistance to starvation and heat stress. Mutations in this region interact epistatically with downstream components of the insulin signaling pathway. Transposon-induced sex-specific and sex-antagonistic effects further accentuate the complex influences that intergenic transposable elements can contribute to quantitative trait phenotypes.     

A conserved developmental program for sensory organ formation in Drosophila melanogaster

Different sensory organs, such the eye and ear, are widely thought to have separate origins, guided by distinct organ-specific factors that direct all aspects of their development. Previous studies of the D. melanogaster gene eyeless (ey) and its vertebrate homolog Pax6 suggested that this gene acts in such a manner and specifically drives eye development. But diverse sensory organs might instead arise by segment-specific modification of a developmental program that is involved more generally in sensory organ formation. In D. melanogaster, a common proneural gene called atonal (ato) functions in the initial process of development of a number of segment-specific organs, including the compound eye, the auditory organ and the stretch receptor, suggesting that these organs share an evolutionary origin. Here we show that D. melanogaster segment-specific sensory organs form through the integration of decapentaplegic (dpp), wingless (wg) and ecdysone signals into a single cis-regulatory element of ato. The induction of ectopic eyes by ey also depends on these signals for ato expression, and the ey mutant eye imaginal disc allows ato expression if cell death is blocked. These results imply that ey does not induce the entire eye morphogenetic program but rather modifies ato-dependent neuronal development. Our findings strongly suggest that various sensory organs evolved from an ato-dependent protosensory organ through segment specification by ey and Hox genes.     

Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster

Loss of cell polarity and cancer are tightly correlated, but proof for a causative relationship has remained elusive. In stem cells, loss of polarity and impairment of asymmetric cell division could alter cell fates and thereby render daughter cells unable to respond to the mechanisms that control proliferation. To test this hypothesis, we generated Drosophila melanogaster larval neuroblasts containing mutations in various genes that control asymmetric cell division and then assayed their proliferative potential after transplantation into adult hosts. We found that larval brain tissue carrying neuroblasts with mutations in raps (also called pins), mira, numb or pros grew to more than 100 times their initial size, invading other tissues and killing the hosts in 2 weeks. These tumors became immortal and could be retransplanted into new hosts for years. Six weeks after the first implantation, genome instability and centrosome alterations, two traits of malignant carcinomas, appeared in these tumors. Increasing evidence suggests that some tumors may be of stem cell origin. Our results show that loss of function of any of several genes that control the fate of a stem cell's daughters may result in hyperproliferation, triggering a chain of events that subverts cell homeostasis in a general sense and leads to cancer.     

A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac

With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the approximately 13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5' regulatory sequences.     

Identification of genes involved in Drosophila melanogaster geotaxis,a complex behavioral trait

Identifying the genes involved in polygenic traits has been difficult. In the 1950s and 1960s, laboratory selection experiments for extreme geotaxic behavior in fruit flies established for the first time that a complex behavioral trait has a genetic basis. But the specific genes responsible for the behavior have never been identified using this classical model. To identify the individual genes involved in geotaxic response, we used cDNA microarrays to identify candidate genes and assessed fly lines mutant in these genes for behavioral confirmation. We have thus determined the identities of several genes that contribute to the complex, polygenic behavior of geotaxis.     

A double-switch system regulates male courtship behavior in male and female Drosophila melanogaster

Current models describe male-specific fruitless (fruM) as a genetic 'switch' regulating sexual behavior in Drosophila melanogaster, and they postulate that female (F) and male (M) doublesex (dsx) products control body sexual morphology. In contradiction to this simple model, we show that dsx, as well as fruM and non-sex-specific retained (retn), affect both male and female sexual behaviors. In females, both retn and dsxF contribute to female receptivity, and both genes act to repress male-like courtship activity in the presence or absence of fruM. In males, consistent with the opposing functions of dsxM and dsxF, dsxM acts as a positive factor for male courtship. retn also acts counter to fruM in the development of the male-specific muscle of Lawrence. Molecularly, retn seems to regulate sexual behavior via a previously described complex that represses zerknullt. Thus, we show that fru and dsx together act as a 'switch' system regulating behavior in the context of other developmental genes, such as retn.