A new brain dopamine-deficient Drosophila and its pharmacological and genetic rescue.

Dopamine (DA) is a neurotransmitter with conserved behavioral roles between invertebrate and vertebrate animals. In addition to its neural functions, in insects DA is a critical substrate for cuticle pigmentation and hardening. Drosophila tyrosine hydroxylase (DTH) is the rate limiting enzyme for DA biosynthesis. Viable brain DA-deficient flies were previously generated using tissue-selective GAL4-UAS binary expression rescue of a DTH null mutation and these flies show specific behavioral impairments. To circumvent the limitations of rescue via binary expression, here we achieve rescue utilizing genomically integrated mutant DTH. As expected, our DA-deficient flies have no detectable DTH or DA in the brain, and show reduced locomotor activity. This deficit can be rescued by l-DOPA/carbidopa feeding, similar to human Parkinson's disease treatment. Genetic rescue via GAL4/UAS-DTH was also successful, although this required the generation of a new UAS-DTH1 transgene devoid of most untranslated regions, as existing UAS-DTH transgenes express in the brain without a Gal4 driver via endogenous regulatory elements. A surprising finding of our newly constructed UAS-DTH1m is that it expresses DTH at an undetectable level when regulated by dopaminergic GAL4 drivers even when fully rescuing DA, indicating that DTH immunostaining is not necessarily a valid marker for DA expression. This finding necessitated optimizing DA immunohistochemistry, showing details of DA innervation to the mushroom body and the central complex. When DA rescue is limited to specific DA neurons, DA does not diffuse beyond the DTH-expressing terminals, such that DA signaling can be limited to very specific brain regions.

The Gene Ontology (GO) database and informatics resource.

The Gene Ontology (GO) project (http://www. geneontology.org/) provides structured, controlled vocabularies and classifications that cover several domains of molecular and cellular biology and are freely available for community use in the annotation of genes, gene products and sequences. Many model organism databases and genome annotation groups use the GO and contribute their annotation sets to the GO resource. The GO database integrates the vocabularies and contributed annotations and provides full access to this information in several formats. Members of the GO Consortium continually work collectively, involving outside experts as needed, to expand and update the GO vocabularies. The GO Web resource also provides access to extensive documentation about the GO project and links to applications that use GO data for functional analyses.

Pairwise RNA structure comparison with stochastic context-free grammars.

Pairwise stochastic context-free grammars ("Pair SCFGs") are powerful tools for finding conserved RNA structures, but unconstrained alignment to Pair SCFGs is prohibitively expensive. We develop versions of the Pair SCFG dynamic programming algorithms that can be conditioned on precomputed structures, significantly reducing the time complexity of alignment. We have implemented these algorithms for general Pair SCFGs in software that is freely available under the GNU Public License.

An expectation maximization algorithm for training hidden substitution models.

We derive an expectation maximization algorithm for maximum-likelihood training of substitution rate matrices from multiple sequence alignments. The algorithm can be used to train hidden substitution models, where the structural context of a residue is treated as a hidden variable that can evolve over time. We used the algorithm to train hidden substitution matrices on protein alignments in the Pfam database. Measuring the accuracy of multiple alignment algorithms with reference to BAliBASE (a database of structural reference alignments) our substitution matrices consistently outperform the PAM series, with the improvement steadily increasing as up to four hidden site classes are added. We discuss several applications of this algorithm in bioinformatics.

An integrated computational pipeline and database to support whole-genome sequence annotation.

We describe here our experience in annotating the Drosophila melanogaster genome sequence, in the course of which we developed several new open-source software tools and a database schema to support large-scale genome annotation. We have developed these into an integrated and reusable software system for whole-genome annotation. The key contributions to overall annotation quality are the marshalling of high-quality sequences for alignments and the design of a system with an adaptable and expandable flexible architecture.

Apollo: a sequence annotation editor.

The well-established inaccuracy of purely computational methods for annotating genome sequences necessitates an interactive tool to allow biological experts to refine these approximations by viewing and independently evaluating the data supporting each annotation. Apollo was developed to meet this need, enabling curators to inspect genome annotations closely and edit them. FlyBase biologists successfully used Apollo to annotate the Drosophila melanogaster genome and it is increasingly being used as a starting point for the development of customized annotation editing tools for other genome projects.

Drosophila fragile X-related gene regulates the MAP1B homolog Futsch to control synaptic structure and function.

Fragile X mental retardation gene (FMR1) encodes an RNA binding protein that acts as a negative translational regulator. We have developed a Drosophila fragile X syndrome model using loss-of-function mutants and overexpression of the FMR1 homolog (dfxr). dfxr nulls display enlarged synaptic terminals, whereas neuronal overexpression results in fewer and larger synaptic boutons. Synaptic structural defects are accompanied by altered neurotransmission, with synapse type-specific regulation in central and peripheral synapses. These phenotypes mimic those observed in mutants of microtubule-associated Futsch. Immunoprecipitation of dFXR shows association with futsch mRNA, and Western analyses demonstrate that dFXR inversely regulates Futsch expression. dfxr futsch double mutants restore normal synaptic structure and function. We propose that dFXR acts as a translational repressor of Futsch to regulate microtubule-dependent synaptic growth and function.

Drosophila neuralized is a ubiquitin ligase that promotes the internalization and degradation of delta.

The Drosophila gene neuralized (neur) has long been recognized to be essential for the proper execution of a wide variety of processes mediated by the Notch (N) pathway, but its role in the pathway has been elusive. In this report, we present genetic and biochemical evidence that Neur is a RING-type, E3 ubiquitin ligase. Next, we show that neur is required for proper internalization of Dl in the developing eye. Finally, we demonstrate that ectopic Neur targets Dl for internalization and degradation in a RING finger-dependent manner, and that the two exist in a physical complex. Collectively, our data indicate that Neur is a ubiquitin ligase that positively regulates the N pathway by promoting the endocytosis and degradation of Dl.

Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays.

To identify new Drosophila genes involved in the immune response, we monitored the gene expression profile of adult flies in response to microbial infection by using high-density oligonucleotide microarrays encompassing nearly the full Drosophila genome. Of 13,197 genes tested, we have characterized 230 induced and 170 repressed by microbial infection, most of which had not previously been associated with the immune response. Many of these genes can be assigned to specific aspects of the immune response, including recognition, phagocytosis, coagulation, melanization, activation of NF-kappaB transcription factors, synthesis of antimicrobial peptides, production of reactive oxygen species, and regulation of iron metabolism. Additionally, we found a large number of genes with unknown function that may be involved in control and execution of the immune response. Determining the function of these genes represents an important challenge for improving our knowledge of innate immunity. Complete results may be found at http://www.fruitfly.org/expression/immunity/.

Joint modeling of DNA sequence and physical properties to improve eukaryotic promoter recognition.

We present an approach to integrate physical properties of DNA, such as DNA bendability or GC content, into our probabilistic promoter recognition system McPROMOTER. In the new model, a promoter is represented as a sequence of consecutive segments represented by joint likelihoods for DNA sequence and profiles of physical properties. Sequence likelihoods are modeled with interpolated Markov chains, physical properties with Gaussian distributions. The background uses two joint sequence/profile models for coding and non-coding sequences, each consisting of a mixture of a sense and an anti-sense submodel. On a large Drosophila test set, we achieved a reduction of about 30% of false positives when compared with a model solely based on sequence likelihoods.

Neuralized is essential for a subset of Notch pathway-dependent cell fate decisions during Drosophila eye development.

Neuralized (neur) is a neurogenic mutant of Drosophila in which many signaling events mediated by the Notch (N) receptor are disrupted. Here, we analyze the role of neur during eye development. Neur is required in a cell-autonomous fashion to restrict R8 and other photoreceptor fates and is involved in lateral inhibition of interommatidial bristles but is not required for induction of the cone cell fate. The latter contrasts with the absolute requirement for Suppressor of Hairless and the Enhancer of split-Complex for cone cell induction. Using gain-of-function experiments, we further demonstrate that ectopic wild-type and truncated Neur proteins can interfere with multiple N-controlled aspects of eye development, including both neur-dependent and neur-independent processes.

neuralized functions cell-autonomously to regulate a subset of notch-dependent processes during adult Drosophila development.

neuralized (neu) represents one of the strong neurogenic mutants in Drosophila. Mutants of this class display, among other phenotypes, a strong overcommitment to neural fates at the expense of epidermal fates. We analyzed the role of neu during adult development by using mutant clonal analysis, misexpression of wild-type and truncated forms of Neu, and examination of genetic interactions with N-pathway mutations. We find that neu is required cell-autonomously for lateral inhibition during peripheral neurogenesis and for multiple asymmetric cell divisions in the sensory lineage. In contrast, neu is apparently dispensable for other N-mediated processes, including lateral inhibition during wing vein development and wing margin induction. Misexpression of wild-type Neu causes defects in both peripheral neurogenesis and wing vein development, while a truncated form lacking the RING finger is further capable of inhibiting formation of the wing margin. In addition, the phenotypes produced by misexpression of wild-type and truncated Neu proteins are sensitive to the dosage of several N-pathway components. Finally, using epitope-tagged Neu proteins, we localize Neu to the plasma membrane and reveal a novel morphology to the sensory organ precursor cells of wing imaginal discs. Collectively, these data indicate a key role for neu in the reception of the lateral inhibitory signal during peripheral neurogenesis.

Transcriptional regulation of cytoskeletal functions and segmentation by a novel maternal pair-rule gene, lilliputian.

Transcriptional control during early Drosophila development is governed by maternal and zygotic factors. We have identified a novel maternal transcriptional regulator gene, lilliputian (lilli), which contains an HMG1 (AT-hook) motif and a domain with similarity to the human fragile X mental retardation FMR2 protein and the AF4 proto-oncoprotein. Embryos lacking maternal lilli expression show specific defects in the establishment of a functional cytoskeleton during cellularization, and exhibit a pair-rule segmentation phenotype. These mutant phenotypes correlate with markedly reduced expression of the early zygotic genes serendipity alpha, fushi tarazu and huckebein, which are essential for cellularization and embryonic patterning. In addition, loss of lilli in adult photoreceptor and bristle cells results in a significant decrease in cell size. Our results indicate that lilli represents a novel pair-rule gene that acts in cytoskeleton regulation, segmentation and morphogenesis.

A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster.

Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the RAS1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of Ras (KSR) functions genetically downstream of RAS1 in this signal transduction cascade. Expression of dominant-negative KSR (KDN) in the developing eye blocks RAS pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate RAS signaling, we screened for genes that alter RAS1/KSR signaling efficiency when misexpressed. In this screen, we recovered three known genes, Lk6, misshapen, and Akap200. We also identified seven previously undescribed genes; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS. We discuss the utility of gain-of-function screens in identifying new components of signaling pathways in Drosophila.

A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in Drosophila.

kinase suppressor of Ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of KSR is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of KSR remains unresolved. To further elucidate the role of KSR and to identify proteins that may be required for KSR function, we conducted a dominant modifier screen in Drosophila based on a KSR-dependent phenotype. Overexpression of the KSR kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with KSR might modify the KSR-dependent phenotype. We screened approximately 185,000 mutagenized progeny for dominant modifiers of the KSR-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for KSR in Ras1 signaling. In addition, we have identified 4 additional complementation groups. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor. The relevance of these loci with respect to the function of KSR and the Ras1 pathway in general is discussed.

The ryanodine receptor is essential for larval development in Drosophila melanogaster.

We have investigated the role of the ryanodine receptor in Drosophila development by using pharmacological and genetic approaches. We identified a P element insertion in the Drosophila ryanodine receptor gene, Ryanodine receptor 44F (Ryr), and used it to generate the hypomorphic allele Ryr(16). An examination of hypodermal, visceral, and circulatory muscle showed that, in each case, muscle contraction was impaired in Ryr(16) larvae. Treatment with the drug ryanodine, a highly specific modulator of ryanodine receptor channel activity, also inhibited muscle function, and, at high levels, completely blocked hypodermal muscle contraction. These results suggest that the ryanodine receptor is required for proper muscle function and may be essential for excitation-contraction coupling in larval body wall muscles. Nonmuscle roles of Ryr were also investigated. Ryanodine-sensitive Ca(2+) stores had previously been implicated in phototransduction; to address this, we generated Ryr(16) mutant clones in the adult eye and performed whole-cell, patch-clamp recordings on dissociated ommatidia. Our results do not support a role for Ryr in normal light responses.

Drosophila p53 binds a damage response element at the reaper locus.

The tumor suppressor gene p53 regulates multiple cellular responses to DNA damage, but the transcriptional targets that specify these responses are incompletely understood. We describe a Drosophila p53 homolog and demonstrate that it can activate transcription from a promoter containing binding sites for human p53. Dominant-negative forms of Drosophila p53 inhibit both transactivation in cultured cells and radiation-induced apoptosis in developing tissues. The cis-regulatory region of the proapoptotic gene reaper contains a radiation-inducible enhancer that includes a consensus p53 binding site. Drosophila p53 can activate transcription from this site in yeast and a multimer of this site is sufficient for radiation induction in vivo. These results indicate that reaper is a direct transcriptional target of Drosophila p53 following DNA damage.