With the completion of genome sequences of major model organisms, increasingly sophisticated genetic tools are necessary for investigating the complex and coordinated functions of genes. Here we describe a genetic manipulation system termed ''genomic engineering'' in Drosophila. Genomic engineering is a 2-step process that combines the ends-out (replacement) gene targeting with phage integrase C31-mediated DNA integration. First, through an improved and modified gene targeting method, a founder knockout line is generated by deleting the target gene and replacing it with an integration site of C31. Second, DNA integration by C31 is used to reintroduce modified target-gene DNA into the native locus in the founder knock-out line. Genomic engineering permits directed and highly efficient modifications of a chosen genomic locus into virtually any desired mutant allele. We have successfully applied the genomic engineering scheme on 6 different genes and have generated at their loci more than 70 unique alleles.cell polarity ͉ ends-out targeting ͉ homologous recombination ͉ phiC31 integrase T he development of homologous recombination (HR)-based gene targeting was a major breakthrough in Drosophila genetics (1, 2). At present, in Drosophila as well as in mice, a HR-based approach is virtually the only way to make directed modifications of a target gene (3, 4). However, because the entire targeting process must be repeated for making each allele, the amount of time and labor may become impractical to make more than just a few targeted alleles. In addition, because of the requirement of HR, it can be very difficult to introduce appreciably complicated DNA sequence modifications by gene targeting. The current lack of adequate genetic tools for directed and efficient modifications of the genome presents a major hurdle in Drosophila genetics today. For example, many of the protein pathways that are highly conserved between Drosophila and vertebrates, such as the cell polarity pathway (5), appear to be exceedingly complex. Rigorous genetic dissections of such intricate protein networks can be highly challenging, because in most cases the functions of mutated or modified individual genes of such pathways can only be assayed by artificial over-expression methods, which often lack the requisite controllability and fidelity of gene expression. One ideal solution would be for each protein gene of interest to generate, at the gene's native genomic locus, a set of defined mutant alleles that are strategically designed to test hypotheses about the protein's in vivo functions and interactions. Furthermore, being able to generate any conceivable alleles of a target gene, such as functional fusion alleles of fluorescent proteins/purification tags or alleles with conditional activities, would also offer us unprecedented freedom and opportunities to explore unique experiments of imaging, proteomics, and disease models.To achieve the goal of such directed, efficient, and versatile modifications of the Drosophila genome, we have developed a...

The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. At the core of the Hippo pathway is a kinase cascade extending from the Hippo (Hpo) tumor suppressor to the Yorkie (Yki) oncoprotein. The Hippo kinase cascade, in turn, is regulated by apical membrane-associated proteins such as the FERM domain proteins Merlin and Expanded (Ex), and the WW- and C2-domain protein Kibra. How these apical proteins are themselves regulated remains poorly understood. Here, we identify the transmembrane protein Crumbs (Crb), a determinant of epithelial apical-basal polarity in Drosophila embryos, as an upstream component of the Hippo pathway in imaginal disk growth control. Loss of Crb leads to tissue overgrowth and target gene expression characteristic of defective Hippo signaling. Crb directly binds to Ex through its juxtamembrane FERM-binding motif (FBM). Loss of Crb or mutation of its FBM leads to mislocalization of Ex to basolateral domain of imaginal disk epithelial cells. These results shed light on the mechanism of Ex regulation and provide a molecular link between apical-basal polarity and tissue growth. Furthermore, our studies implicate Crb as a putative cell surface receptor for Hippo signaling by uncovering a transmembrane protein that directly binds to an apical component of the Hippo pathway.

Highlights d The authors propose a new concept of the chemoconnectome (CCT) for chemical transmission d Chemoconnectomics with genetic tools is a new approach for neural mapping d CCT research in Drosophila will stimulate CCT studies in higher animals

Our understanding of the molecular mechanisms underlying sleep homeostasis is limited. We have taken a systematic approach to study neural signaling by the transmitter 5-hydroxytryptamine (5-HT) in drosophila. We have generated knockout and knockin lines for Trh, the 5-HT synthesizing enzyme and all five 5-HT receptors, making it possible for us to determine their expression patterns and to investigate their functional roles. Loss of the Trh, 5HT1a or 5HT2b gene decreased sleep time whereas loss of the Trh or 5HT2b gene diminished sleep rebound after sleep deprivation. 5HT2b expression in a small subset of, probably a single pair of, neurons in the dorsal fan-shaped body (dFB) is functionally essential: elimination of the 5HT2b gene from these neurons led to loss of sleep homeostasis. Genetic ablation of 5HT2b neurons in the dFB decreased sleep and impaired sleep homeostasis. Our results have shown that serotonergic signaling in specific neurons is required for the regulation of sleep homeostasis.

Dysregulation of eating behavior can lead to obesity, which affects 10% of the adult population worldwide and accounts for nearly 3 million deaths every year. Despite this burden on society, we currently lack effective pharmacological treatment options to regulate appetite. We used Drosophila melanogaster larvae to develop a high-throughput whole organism screen for drugs that modulate food intake. In a screen of 3630 small molecules, we identified the serotonin (5-hydroxytryptamine or 5-HT) receptor antagonist metitepine as a potent anorectic drug. Using cell-based assays we show that metitepine is an antagonist of all five Drosophila 5-HT receptors. We screened fly mutants for each of these receptors and found that serotonin receptor 5-HT2A is the sole molecular target for feeding inhibition by metitepine. These results highlight the conservation of molecular mechanisms controlling appetite and provide a method for unbiased whole-organism drug screens to identify novel drugs and molecular pathways modulating food intake.