Rachael French

To identify genes expressed in interesting spatial and temporal patterns during development, we mobilized transposons carrying the yeast transcriptional activator, GAL4, to create new insertions throughout the Drosophila genome. At these new sites, neighboring enhancers drive expression of GAL4 in a pattern similar to that of nearby genes (Brand and Perrimon, 1993). Since GAL4 binds to a UAS target sequence, flies containing a GAL4 transposon can activate transcription of a UAS-linked gene, thereby resulting in controlled expression of that secondary gene. Thus, the GAL4 P element is a useful tool both for identifying genes of interest and for ectopically expressing genes in novel tissues or at specific developmental stages. We mobilized a GAL4 insertion on the second chromosome (P{GawB} CY2; Queenan et al., 1997) to generate a collection of GAL4-expressing enhancer trap lines. F1 “jumpstarter” males (w/Y; GAL4/CyO; Sb 2-3/ ) were mated in vials to attached-X (C(1)DX/Y) females. From 200 such crosses, red-eyed, curly-winged, long-bristled sons (bearing a new GAL4 insertion) were selected and mated individually to w/w females to establish 50 lines. Chromosome-mapping data, enhancer trap pattern, and other phenotypic characters demonstrated that four insertions were duplicates, yielding a total of 46 independent lines (Table 1). All X and third chromosome lines are viable, although three lines exhibit reduced viability or fertility (lines 15, 45, and 50). We could not determine the viability of the CyOlinked lines since the balancer chromosome is homozygous lethal. The genotypes of the resulting stocks are: X-chromosome lines, w P{GawB}, second-chromosome lines, y w; Pin/CyO, P{GawB}, and third chromosome lines, y w; III P{GawB}. Line 15 also carries the FM6, y w Bar balancer while lines 45 and 50 contain TM3, Sb Ser. Finally, line number three exhibits a striking eye phenotype consisting of a variable loss of eye pigment in a gradient along the anterior/posterior axis. Surprisingly, this line lacks detectable GAL4 expression in larval eye discs. We characterized sequences flanking a subset of the insertions in our collection by the inverse PCR method (http:www.fruitfly.org/methods/). Table 1 lists neighboring predicted or known genes. To identify and visualize the morphology of cells expressing GAL4, we crossed females from each line to males carrying UAS-taulacZ, which links the -galactosidase enzyme to the tubulin-binding protein Tau (Hildago et al., 1995). Some lines (1, 7, 10 (males only), 14, 22, 25, 26, 32, 35, 36, and 41) were lethal in pupal stages when crossed to this reporter; therefore, to assay ovary and testis expression, we crossed these strains to UAS-lacZ.NZ, which contains a nuclear-localization signal (Y. Hiromi and S. West, unpubl. results). Tables 1 and 2 describe the expression patterns for each line and Figure 1 shows selected patterns that illustrate key points. More detailed descriptions of all the patterns are available upon request. All lines exhibit interesting staining in at least one developmental stage. As commonly noted, we observe patchy or variable GAL4 expression in many lines. For example, GAL4 drives embryonic PNS expression in 18 lines (Table 1). While all embryos within a given strain exhibit a general PNS pattern that is consistent, within an individual embryo, slight variations often occur between segmental repeats (e.g., brackets in Fig. 1a). Only a few lines express GAL4 in a single tissue at a specific developmental stage. For example, line 20 drives expression in the longitudinal visceral mesoderm from stage 12 onwards (Fig. 1c). When crossed to UASGFP, line 20 can be used to visualize this highly migratory tissue in vivo. As Tables 1 and 2 indicate, many lines express GAL4 in identical tissues. Nevertheless, important differences may exist in the distribution of expression within each tissue. For example, Fig. 1f–i shows representative wing discs from four lines that express GAL4 in all imaginal discs, the brain, and the central nervous system (IGD, brain, and CNS). The distinct patterns shown in the wing discs presumably reveal specific regulatory elements that govern expression of nearby

The Drosophila gene tramtrack (ttk) encodes two transcriptional repressors, Ttk69 and Ttk88, which are required for normal embryogenesis and imaginal disc development. Here, we characterize a novel female sterile allele of tramtrack called twin peaks (ttk(twk)) that, unlike othertramtrack alleles, has no effect on viability and produces no obvious morphological defects, except during oogenesis. Females homozygous for twin peaks produce small eggs with thin eggshells and short dorsal respiratory appendages. Complementation analyses, immunolocalization, and rescue data demonstrate that these defects are due to loss of Ttk69, which is expressed in the follicle cells and is required for normal chorion production and dorsal follicle-cell migration. Analyses of phenotypes produced by mutations in other loci that regulate eggshell synthesis suggest that the chorion production and follicle-cell migration defects are independent. We present evidence that twin peaks disrupts a promoter or promoters required for late-stage follicle-cell expression of Ttk69. We hypothesize that loss of Ttk69 in all follicle cells disrupts chorion gene expression and lack of function in dorsal anterior follicle cells inhibits morphogenetic changes required for elongating the dorsal appendages.

The function of an organ relies on its form, which in turn depends on the individual shapes of the cells that create it and the interactions between them. Despite remarkable progress in the field of developmental biology, how cells collaborate to make a tissue remains an unsolved mystery. To investigate the mechanisms that determine organ structure, we are studying the cells that form the dorsal appendages (DAs) of the Drosophila melanogaster eggshell. These cells consist of two differentially patterned subtypes: roof cells, which form the outward-facing roof of the lumen, and floor cells, which dive underneath the roof cells to seal off the floor of the tube. In this paper, we present three lines of evidence that reveal a further stratification of the DA-forming epithelium. Laser ablation of only a few cells in the anterior of the region causes a disproportionately severe shortening of the appendage. Genetic alteration through the twin peaks allele of tramtrack69 (ttk(twk)), a female-sterile mutation that leads to severely shortened DAs, causes no such shortening when removed from a majority of the DA-forming cells, but rather, produces short appendages only when removed from cells in the very anterior of the tube-forming tissue. Additionally we show that heterotrimeric G-protein function is required for DA morphogenesis. Like TTK69, Gbeta 13F is not required in all DA-forming follicle cells but only in the floor and leading roof cells. The different phenotypes that result from removal of Gbeta 13F from each region demonstrate a striking division of function between different DA-forming cells. Gbeta mutant floor cells are unable to control the width of the appendage while Gbeta mutant leading roof cells fail to direct the elongation of the appendage and the convergent-extension of the roof-cell population.

Understanding how genes can direct behaviors has been a prevailing goal of neuroscience. Courtship in D. melanogaster is a complex yet stereotyped array of behaviors established by sex-specific genetic pathways mediated by sensory inputs by the nervous system. However, much remains to be discovered about the neurobiological and molecular mechanisms that regulate this complex set of behaviors. We have identified a group of cells expressing Trapped in endoderm 1 (Tre1) in which male Fruitless proteins are required to reduce the speed of courtship initiation. Tre1 encodes a G-protein-coupled receptor required for establishment of cell polarity and cell migration and has previously not been shown to be involved in courtship behavior. By monitoring the latency to courtship initiation of male flies, we found the expression of female-specific transcription factors in Tre1-expressing neurons, or "feminization," resulted in rapid courtship initiation. The Tre1-feminized males produ...

Alcohol is a known teratogen, and developmental exposure to ethanol results in Fetal Alcohol Spectrum Disorder (FASD). Children born with FASD can exhibit a range of symptoms including low birth weight, microcephaly, and neurobehavioral problems. Treatment of patients with FASD is estimated to cost 4 billion dollars per year in the United States alone, and 2 million dollars per affected individual's lifetime. We have established Drosophila melanogaster as a model organism for the study of FASD. Here we report that mutations in Dementin (Dmtn), the Drosophila ortholog of the Alzheimer Disease associated protein TMCC2, convey sensitivity to developmental ethanol exposure, and provide evidence that Dmtn expression is disrupted by ethanol. In addition, we find that flies reared on ethanol exhibit mild climbing defects suggestive of neurodegeneration. Surprisingly, our data also suggest that flies reared on ethanol age more slowly than control animals, and we find that a number of sl...

Understanding the role of genes in directing behavior is one of the primary goals of neuroscience. Mating behavior in Drosophila is controlled by male-specific splicing of the master regulatory gene fruitless (fru), and the male-specific splice form, fruM, is both necessary and sufficient for all aspects of the courtship ritual. We have previously described the role of Trapped in endoderm 1 (Tre1) in courtship behavior. Tre1 encodes an orphan G-protein-coupled receptor that is essential for normal courtship behavior in male flies. We previously found that feminizing Tre1-expressing cells in males via expression of the female-specific splicing factor Transformer (TraF) resulted in rapid courtship initiation. Here we confirm that Tre1 is required in neurons for normal courtship behavior, and present genetic evidence that Tre1 acts through the downregulation of the E-cadherin Shotgun, and that the neurotransmitter histamine is the likely Tre1 ligand. Our findings are the first evidence...

Over the past 35 years, developmental geneticists have made impressive progress towards an understanding of how genes specify morphology and function, particularly as relates to the specification of each physical component of an organism. In the last 20 years, male courtship behavior in Drosophila melanogaster has emerged as a robust model system for the study of genetic specification of behavior. Courtship behavior is both complex and innate, and a single gene, fruitless (fru), is both necessary and sufficient for all aspects of the courtship ritual. Typically, loss of male-specific Fruitless proteins function results in male flies that perform the courtship ritual incorrectly, slowly, or not at all. Here we describe a novel requirement for fru: we have identified a group of cells in which male Fru proteins are required to reduce the speed of courtship initiation. In addition, we have identified a gene, Trapped in endoderm 1 (Tre1), which is required in these cells for normal court...

Fetal Alcohol Syndrome (FAS) is caused by alcohol consumption during pregnancy and affects 1-3 live births per 1000 in the U.S. FAS individuals exhibit phenotypes that include decreased survival, developmental delay, growth deficits, abnormal response to ethanol exposure, behavioral problems, and cognitive deficits. These cognitive deficits are among the most socially consequential aspects of FAS. We have previously shown that rearing Drosophila melanogaster on ethanol-treated food during larval development results in FAS-like phenotypes. This is consistent with studies that have demonstrated homology in Drosophila and vertebrate responses to ethanol. Cognitive performance, however, has not been studied in ethanol-reared flies. To expand the FAS model, this study introduces cognitive assessment of ethanol-reared flies via learning and memory assays. We hypothesize that ethanol-reared flies will exhibit defects in learning and memory relative to control flies. From the battery of est...

Thesis (Ph. D.)--University of Washington, 2003 The Drosophila gene tramtrack (ttk) encodes two transcriptional repressors, Ttk69 and Ttk88, which are required for normal embryogenesis and imaginal disc development. Here I characterize a novel female sterile allele of tramtrack called twin peaks (ttktwk) that, unlike other tramtrack alleles, has no effect on viability and produces no obvious morphological defects, except during oogenesis. Females homozygous for twin peaks produce small eggs with thin eggshells and short dorsal respiratory appendages. Complementation analyses, immunolocalization and rescue data demonstrate that these defects are due to loss of Ttk69, which is expressed in the follicle cells and is required for normal chorion production and dorsal follicle cell migration. Analyses of phenotypes produced by mutations in other loci that regulate eggshell synthesis suggest that the chorion production and follicle cell migration defects are independent. I present evidence...

Drosophila females lay eggs in rotting fruit, resulting in larval exposure to high concentrations of ethanol. These ethanol concentrations are toxic, leading to significant lethality and developmental delays. Our previous work demonstrates that these phenotypes are partially mediated by the insulin receptor. The insulin receptor can activate several pathways, including the Ras/Mitogen Activated Protein Kinase (MAPK) pathway, which is important in cell growth, differentiation, and proliferation. Unpublished data from our lab indicates that increasing activity of this pathway is protective against the toxic effects of ethanol. The goal of this project is to further investigate the role of the MAPK pathway in the developmental response to ethanol. We predict that increasing MAPK pathway activity will result in protection, while reduced activity will lead to ethanol sensitivity. As predicted, ubiquitous expression of an RNA interference (RNAi) construct targeting vacuolar peduncles (vap...

Fetal alcohol syndrome (FAS) is a spectrum disorder affecting individuals exposed to ethanol during gestation and often results in developmental delays, decreased survival rates, growth and development defects, behavioral changes, and altered adult responses to ethanol; it is also the leading cause of non-genetic mental retardation. Previous studies have shown Drosophila melanogaster larvae exposed to ethanol-treated food model these phenotypes. The purpose of this study is to determine whether oxidative stress is playing a role in these ethanol-induced phenotypes. We hypothesize that artificially inducing oxidative stress should phenocopy some or all of the FAS symptoms, while alleviating oxidative stress should correspondingly ameliorate the phenotypes. Both pharmacological and genetic manipulations were utilized to induce or alleviate oxidative stress, either alone, or in conjunction with ethanol exposure. To date, using transgenic constructs to pan-neuronally upregulate the anti...

Signal transduction pathways are sensitive to environmental stresses, and oxidative stress is a fundamental process nearly all organisms experience. Drosophila melanogaster reared in ethanol-treated food have reduced viability and developmental delays, among other phenotypes. Previous work has shown that upregulation of insulin production can rescue these phenotypes, and unpublished data from the French lab implicates the PI3K pathway in mediating ethanol-induced lethality. Given that ethanol is known to induce oxidative stress, and the insulin pathway is known to mediate oxidative damage, the goal of this research project is to evaluate the interactions of increased oxidative stress and ethanol on the survival and developmental delay of larvae reared in ethanol and paraquat treated food. Our hypothesis is that at least some of the ethanol-induced phenotypes are a result of oxidative stress and that combining ethanol exposure with a known oxidative stress-inducing agent will produce...

Ethanol exposure during development causes an array of developmental abnormalities, both physiological and behavioral. In mammals, these abnormalities are collectively known as fetal alcohol effects (FAE) or fetal alcohol spectrum disorder (FASD). We have established a Drosophila melanogaster model of FASD and have previously shown that developmental ethanol exposure in flies leads to reduced expression of insulin-like peptides (dILPs) and their receptor. In this work, we link that observation to dysregulation of fatty acid metabolism and lipid accumulation. Further, we show that developmental ethanol exposure in Drosophila causes oxidative stress, that this stress is a primary cause of the developmental lethality and delay associated with ethanol exposure, and, finally, that one of the mechanisms by which ethanol increases oxidative stress is through abnormal fatty acid metabolism. These data suggest a previously uncharacterized mechanism by which ethanol causes the symptoms associ...

It has long been known that heavy alcohol consumption leads to neuropathology and neuronal death. While the response of neurons to an ethanol insult is strongly influenced by genetic background, the underlying mechanisms are poorly understood. Here, we show that even a single intoxicating exposure to ethanol causes non-cell-autonomous apoptotic death specifically of Drosophila olfactory neurons, which is accompanied by a loss of a behavioral response to the smell of ethanol and a blackening of the third antennal segment. The Drosophila homolog of glycogen synthase kinase-3 (GSK-3)β, Shaggy, is required for ethanol-induced apoptosis. Consistent with this requirement, the GSK-3β inhibitor lithium protects against the neurotoxic effects of ethanol, indicating the possibility for pharmacological intervention in cases of alcohol-induced neurodegeneration. Ethanol-induced death of olfactory neurons requires both their neural activity and functional NMDA receptors. This system will allow t...

Page 1. WPM 3.3: A 2ns Cycle, 4ns Access 512kb CMOS ECL SRAM Terry I. Chappell, Barbara A. Chappell, Stanley E. Schuster, James W. Allan, Stephen P. Klepner, Rajiv V. Jashi, Robert L. Franch IBM T. J. Watson Research Center, YorMown Heights, NY ...

Fetal Alcohol Syndrome (FAS) is a spectrum disorder affecting individuals exposed to ethanol during gestation and results in growth and development defects, behavioral changes, and altered adult responses to ethanol. We have previously demonstrated that Drosophila melanogaster larvae reared in ethanol-containing food display many of the same phenotypes. We have also shown that upregulation of insulin during development rescues many of these ethanol-induced phenotypes. The purpose of this study is to determine whether downstream components of the phosphoinositide 3-kinase (PI3K) pathway are mediating these phenotypes. Flies reared in 5-7% ethanol show reduced survival (approximately 50% of control survival). If the PI3K pathway is involved in mediating ethanol-induced phenotypes, then decreased pathway activity will amplify this effect, while increased activity should result in increased survival relative to wildtype flies. We therefore generated flies with altered levels of PI3K sig...

We are investigating the cellular mechanisms by which oxidative damage leads to developmental alterations, as well as whether ethanol’s developmental effects are through an increase in oxidative stress. Our previously published results show that developmental exposure to ethanol in Drosophila melanogaster leads to reduced viability and a significant developmental delay, and that these phenotypes can be ameliorated with transgenic expression of Drosophila insulin-like proteins (Dilps) (French, McClure, and Heberlein, 2011). Previous research in our lab indicates that mutation of the fly homolog of PDK in the PI3K insulin-signaling pathway suppresses ethanol-induced developmental lethality. This mutation has previously been shown to lead to resistance to oxidative stress. This fact, combined with the fact that ethanol is known to cause oxidative stress, led us to hypothesize that oxidative stress might be at least partially responsible for the ethanol-induced phenotypes. To test this ...

Reactive oxygen species (ROS) are unstable oxygen-containing molecules that oxidize most cellular macromolecules. Because ROS occur as a natural result of cellular metabolism, they are experienced by all aerobically respiring organisms. Ethanol is known to increase the production of reactive oxygen species (ROS) in a variety of ways, leading to oxidative stress. Drosophila melanogaster exposed to ethanol during development have decreased survival and a significant developmental delay. Our lab has unpublished data indicating that oxidative stress can phenocopy the effects of ethanol exposure. We hypothesize that at least some of ethanol’s toxic effects are due to oxidative stress. One way to test this hypothesis is to test ethanol and peroxide for synergistic effects on fly development. We will rear flies on a combination of ethanol and hydrogen peroxide, with each chemical at a sublethal concentration. If the delay and lethality caused by ethanol is due to oxidative stress, we expec...

Prenatal exposure to ethanol in humans results in a wide range of developmental abnormalities, including growth deficiency, developmental delay, reduced brain size, permanent neurobehavioral abnormalities and fetal death. Here we describe the use of Drosophila melanogaster as a model for exploring the effects of ethanol exposure on development and behavior. We show that developmental ethanol exposure causes reduced viability, developmental delay and reduced adult body size. We find that flies reared on ethanol-containing food have smaller brains and imaginal discs, which is due to reduced cell division rather than increased apoptosis. Additionally, we show that, as in mammals, flies reared on ethanol have altered responses to ethanol vapor exposure as adults, including increased locomotor activation, resistance to the sedating effects of the drug and reduced tolerance development upon repeated ethanol exposure. We have found that the developmental and behavioral defects are largely ...