Genetic Annotation of Gain-Of-Function Screens Using RNA Interference and in Situ Hybridization of Candidate Genes in the Drosophila Wing

Gain-of-function screens in Drosophila are an effective method with which to identify genes that affect the development of particular structures or cell types. It has been found that a fraction of 2–10% of the genes tested, depending on the particularities of the screen, results in a discernible phenotype when overexpressed. However, it is not clear to what extent a gain-of-function phenotype generated by overexpression is informative about the normal function of the gene. Thus, very few reports attempt to correlate the loss- and overexpression phenotype for collections of genes identified in gain-of-function screens. In this work we use RNA interference and in situ hybridization to annotate a collection of 123 P-GS insertions that in combination with different Gal4 drivers affect the size and/or patterning of the wing. We identify the gene causing the overexpression phenotype by expressing, in a background of overexpression, RNA interference for the genes affected by each P-GS insertion. Then, we compare the loss and gain-of-function phenotypes obtained for each gene and relate them to its expression pattern in the wing disc. We find that 52% of genes identified by their overexpression phenotype are required during normal development. However, only in 9% of the cases analyzed was there some complementarity between the gain- and loss-of-function phenotype, suggesting that, in general, the overexpression phenotypes would not be indicative of the normal requirements of the gene.     

A Genetic Analysis of Deltex and Its Interaction with the Notch Locus in Drosophila Melanogaster

During Drosophila development networks of genes control the developmental pathways that specify cell fates. The Notch gene is a well characterized member of some cell fate pathways, and several other genes belonging to these same pathways have been identified because they share a neurogenic null phenotype with Notch. However, it is unlikely that the neurogenic genes represent all of the genes in these pathways. The goal of this research was to use a genetic approach to identify and characterize one of the other genes that acts with Notch to specify cell fate. Mutant alleles of genes in the same pathway should have phenotypes similar to Notch alleles and should show phenotypic interactions with Notch alleles. With this approach we identified the deltex gene as a potential cell fate gene. An extensive phenotypic characterization of loss-of-function deltex phenotypes showed abnormalities (such as thick wing veins, double bristles and extra cone cells) that suggest that deltex is involved in cell fate decision processes. Phenotypic interactions between deltex and Notch as seen in double mutants showed that Notch and deltex do not code for duplicate functions and that the two genes function together in many different developing tissues. The results of these investigations lead to the conclusion that the deltex gene functions with the Notch gene in one or more developmental pathways to specify cell fate.     

Drosophila melanogaster chemosensory and muscle development: Identification and properties of a novel allele ofscalloped and of a new locus, SG18.1, in a Gal4 enhancer trap screen

Our primary interest is to probe into the genetic and molecular mechanisms underlying the development of the chemosensory and neuromuscular systems inDrosophila melanogaster. We have generated and characterized 40 Gal4 enhancer trap lines with P-Gal4 insertion as an attempt to identify genes with a likely role in the development and differentiation of chemosensory and neuromuscular tissues, and at the same time to obtain Gal4 drivers that would facilitate targeted ectopic expression of genes in these tissues. Insertion strain SG18.1 has reporter gene activity in major olfactory components of the adult fly and in their presumptive areas in the imaginal discs. SG29.1 has an insertion in thescalloped gene and has been useful in understanding genetic interactions that pattern the wing and in defining the role ofscalloped in muscle development in flies.     

Son of Notch, a winged-helix gene involved in boundary formation in the Drosophila wing

A P element enhancer trap screen was conducted to identify genes involved in dorsal-ventral boundary formation in Drosophila. The son of Notch (son) gene was identified by the son(2205) enhancer trap insertion, which is a partial loss-of-function mutation. Based on son(2205) mutant phenotypes and genetic interactions with Notch and wingless mutations, we conclude that son participates in wing development, and functions in the Notch signaling pathway at the dorsal-ventral boundary in the wing. Notch signaling pathway components activate son enhancer trap expression in wing cells. son enhancer trap expression is regulated positively by wingless, and negatively by cut in boundary cells. Ectopic Son protein induces wingless and cut expression in wing discs. We hypothesize that there is positive feedback regulation of son by wingless, and negative regulation by cut at the dorsal-ventral boundary during wing development.     

Extramacrochaetae,a trans-acting gene of the achaete-scute complex of Drosophila involved in cell communication

Summary Phenotypic analyses of genetic combinations involving the gene extramacrochaetae (emc) reveal its participation in the differentiation of both sensory elements and wing veins. The study of near-amorphic alleles of emc in mitotitc recombination clones indicates that it also affects cell proliferation. These clones show abnormal sizes, shapes and spatial distribution. They differentiate extra sensory elements as well as extra veins. A gain of function mutation in the gene causes opposite phenotypes in both differentiation systems. The effects of the mutant on proliferation and patterning are consistent with the emc gene being involved in the transfer of information between neighbouring cells, which leads to the spatial expression of the achaetescute gene complex and genes involved in vein formation.     

Mutations modulating the Argos-regulated signaling pathway in Drosophila eye development

Argos is a secreted protein that contains an EGF-like domain and acts as an inhibitor of Drosophila EGF receptor activation. To identify genes that function in the Argos-regulated signaling pathway, we performed a genetic screen for enhancers and suppressors of the eye phenotype caused by the overexpression of argos. As a result, new alleles of known genes encoding components of the EGF receptor pathway, such as Star, sprouty, bulge, and clown, were isolated. To study the role of clown in development, we examined the eye and wing phenotypes of the clown mutants in detail. In the eye discs of clown mutants, the pattern of neuronal differentiation was impaired, showing a phenotype similar to those caused by a gain-of-function EGF receptor mutation and overexpression of secreted Spitz, an activating ligand for the EGF receptor. There was also an increased number of pigment cells in the clown eyes. Epistatic analysis placed clown between argos and Ras1. In addition, we found that clown negatively regulated the development of wing veins. These results suggest that the clown gene product is important for the Argos-mediated inhibition of EGF receptor activation during the development of various tissues. In addition to the known genes, we identified six mutations of novel genes. Genetic characterization of these mutants suggested that they have distinct roles in cell differentiation and/or survival regulated by the EGF receptor pathway.     

A gain-of-function screen in zebrafish identifies a guanylate cyclase with a role in neuronal degeneration

Manipulation of gene expression is one of the most informative ways to study gene function. Genetic screens have been an informative method to identify genes involved in developmental processes. In the zebrafish, loss-of-function screens have been the primary approach for these studies. We sought to complement loss-of-function screens using an unbiased approach to overexpress genes with a Gal4-UAS based system, similar to the gain-of-function screens in Drosophila. Using MMLV as a mutagenic vector, a cassette containing a UAS promoter was readily inserted in the genome, often at the 5′ end of genes, allowing Gal4-dependent overexpression. We confirmed that genes downstream of the viral insertions were overexpressed in a Gal4-VP16 dependent manner. We further demonstrate that misexpression of one such downstream gene gucy2F, a membrane-bound guanylate cyclase, throughout the nervous system results in multiple defects including a loss of forebrain neurons. This suggests proper control of cGMP production is important in neuronal survival. From this study, we propose that this gain-of-function approach can be applied to large-scale genetic screens in a vertebrate model organism and may reveal previously unknown gene function. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. FJ151012, FJ151013, and FJ151014.     

MKP-3 has essential roles as a negative regulator of the Ras/mitogen-activated protein kinase pathway during Drosophila development

Mitogen-activated protein kinase (MAPK) phosphatase 3 (MKP-3) is a well-known negative regulator in the Ras/extracellular signal-regulated kinase (ERK)-MAPK signaling pathway responsible for cell fate determination and proliferation during development. However, the physiological roles of MKP-3 and the mechanism by which MKP-3 regulates Ras/Drosophila ERK (DERK) signaling in vivo have not been determined. Here, we demonstrated that Drosophila MKP-3 (DMKP-3) is critically involved in cell differentiation, proliferation, and gene expression by suppressing the Ras/DERK pathway, specifically binding to DERK via the N-terminal ERK-binding domain of DMKP-3. Overexpression of DMKP-3 reduced the number of photoreceptor cells and inhibited wing vein differentiation. Conversely, DMKP-3 hypomorphic mutants exhibited extra photoreceptor cells and wing veins, and its null mutants showed striking phenotypes, such as embryonic lethality and severe defects in oogenesis. All of these phenotypes were highly similar to those of the gain-of-function mutants of DERK/rl. The functional interaction between DMKP-3 and the Ras/DERK pathway was further confirmed by genetic interactions between DMKP-3 loss-of-function mutants or overexpressing transgenic flies and various mutants of the Ras/DERK pathway. Collectively, these data provide the direct evidences that DMKP-3 is indispensable to the regulation of DERK signaling activity during Drosophila development.     

Enhancers of Glp-1, a Gene Required for Cell-Signaling in Caenorhabditis Elegans, Define a Set of Genes Required for Germline Development

The distal tip cell (DTC) regulates the proliferation or differentiation choice in the Caenorhabditis elegans germline by an inductive mechanism. Cell signaling requires a putative receptor in the germline, encoded by the glp-1 gene, and a putative signal from the DTC, encoded by the lag-2 gene. Both glp-1 and lag-2 belong to multigene gene families whose members are essential for cell signaling during development of various tissues in insects and vertebrates as well as C. elegans. Relatively little is known about how these pathways regulate cell fate choice. To identify additional genes involved in the glp-1 signaling pathway, we carried out screens for genetic enhancers of glp-1. We recovered mutations in five new genes, named ego (enhancer of glp-1), and two previously identified genes, lag-1 and glp-4, that strongly enhance a weak glp-1 loss-of-function phenotype in the germline. Ego mutations cause multiple phenotypes consistent with the idea that gene activity is required for more than one aspect of germline and, in some cases, somatic development. Based on genetic experiments, glp-1 appears to act upstream of ego-1 and ego-3. We discuss the possible functional relationships among these genes in light of their phenotypes and interactions with glp-1.     

Genetic characterization of the Drosophila melanogaster Suppressor of deltex gene: A regulator of notch signaling.

The Notch receptor signaling pathway regulates cell differentiation during the development of multicellular organisms. A number of genes are known to be components of the pathway or regulators of the Notch signal. One candidate for a modifier of Notch function is the Drosophila Suppressor of deltex gene [Su(dx)]. We have isolated four new alleles of Su(dx) and mapped the gene between 22B4 and 22C2. Loss-of-function Su(dx) mutations were found to suppress phenotypes resulting from loss-of-function of Notch signaling and to enhance gain-of-function Notch mutations. Hairless, a mutation in a known negative regulator of the Notch pathway, was also enhanced by Su(dx). Phenotypes were identified for Su(dx) in wing vein development, and a role was demonstrated for the gene between 20 and 30 hr after puparium formation. This corresponds to the period when the Notch protein is involved in refining the vein competent territories. Taken together, our data indicate a role for Su(dx) as a negative regulator of Notch function.