dissection-and-mounting-of-drosophila-pupal-eye-discs

Dissection and Mounting of <em>Drosophila</em> Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Midwestern University

Activation of the luteinizing hormone beta (LHbeta) promoter by gonadotropin-releasing hormone (GnRH) via the transcription factor early growth response protein-1 (Egr1) has been well characterized. To determine the mechanisms affecting Egr1 regulation of LHbeta, we analyzed five different species of LHbeta promoters (equine, mouse, rat, bovine and human). Electrophoretic mobility shift assays (EMSAs) identified multiple transcription factors binding to the Egr regions on the LHbeta promoter. Species-specific differences existed in the binding affinity for Sp1, Sp3, steroidogenic factor-1 (SF-1) and Egr1. Upon mutation of the Egr elements, competition for the binding of all zinc finger proteins was lost, suggesting that the Sp proteins compete for binding to the same site that Egr1 occupies. In addition, the promoters from species that had the highest affinity for Sp1 also had the lowest activation by Egr1 and GnRH. Thus we hypothesize that Sp1 competes for Egr1 binding to the Egr elements on the LHbeta promoter and thus inhibits the ability of GnRH and Egr1 to activate the LHbeta promoter.

Species differences in GnRH activation of the LHbeta promoter: role of Egr1 and Sp1.

Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers.

We conducted a screen for glossy-eye flies that fail to incorporate BrdU in the third larval instar eye disc but exhibit normal neuronal differentiation and isolated 23 complementation groups of mutants. These same phenotypes were previously seen in mutants for cytochrome c oxidase subunit Va. We have molecularly characterized six complementation groups and, surprisingly, each encodes a mitochondrial protein. Therefore, we believe our screen to be an efficient method for identifying genes with mitochondrial function.

An efficient genetic screen in Drosophila to identify nuclear-encoded genes with mitochondrial function.

Stem cells, which can self-renew and generate differentiated cells, have been shown to be controlled by surrounding microenvironments or niches in several adult tissues. However, it remains largely unknown what constitutes a functional niche and how niche formation is controlled. In the Drosophila ovary, germline stem cells (GSCs), which are adjacent to cap cells and two other cell types, have been shown to be maintained in the niche. In this study, we show that Notch signaling controls formation and maintenance of the GSC niche and that cap cells help determine the niche size in the Drosophila ovary. Expanded Notch activation causes the formation of more cap cells and bigger niches, which support more GSCs, whereas compromising Notch signaling during niche formation decreases the cap cell number and niche size and consequently the GSC number. Furthermore, the niches located away from their normal location can still sufficiently sustain GSC self-renewal by maintaining high local BMP signaling and repressing bam as in normal GSCs. Finally, loss of Notch function in adults results in rapid loss of the GSC niche, including cap cells and thus GSCs. Our results indicate that Notch signaling is important for formation and maintenance of the GSC niche, and that cap cells help determine niche size and function.

Notch signaling controls germline stem cell niche formation in the Drosophila ovary.

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes.

Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes.

Annual Drosophila research conference, 2008.

We combined Gal4-UAS and the FLP recombinase-FRT and fluorescent reporters to generate cell clones that provide spatial, temporal and genetic information about the origins of individual cells in Drosophila melanogaster. We named this combination the Gal4 technique for real-time and clonal expression (G-TRACE). The approach should allow for screening and the identification of real-time and lineage-traced expression patterns on a genomic scale.

G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila.

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Shop talk: Annual Drosophila Research Conference, 2010.

Shop talk : Congr&egrave;s annuel de recherche de drosophile, 2010.

Shop Talk: j&auml;hrliche Drosophila Research Conference, 2010.

Shop talk: conferenza annuale della drosofila ricerca, 2010.

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Hablar de la tienda: Conferencia anual de investigaci&oacute;n de Drosophila, 2010.

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Anual Conferencia de investigaci&oacute;n de Drosophila, 2011.

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Annual Drosophila research conference, 2011.

Drosophila Congr&egrave;s annuel de recherche, 2011.

Drosophila Forschung Jahreskonferenz, 2011.

Drosofila ricerca Conferenza annuale 2011.

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Annual Drosophila Research Conference, 2012.

Drosophila Annual Research Conference, 2012.

Drosophila Forschung Jahreskonferenz, 2012.

Drosofila ricerca Conferenza annuale, 2012.

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Anual Conferencia de investigaci&oacute;n de Drosophila, 2012.

The mechanism by which inhaled anesthetics work is not fully understood, although they have been extensively used. Much research has been done showing the likelihood that there is more than one pathway or mechanism of action. A long-term goal of our laboratory is to decipher these mechanisms using Drosophila melanogaster, an excellent model organism for this purpose. In order to do this, we have modified and constructed an apparatus called the inebriometer to quantitatively analyze the response of flies to inhaled anesthetics. While the inebriometer is not new to the fly community, our updated design provides a relatively low-labor, high-throughput means for performing screens in search of genes involved in the anesthetic mechanism. Here we describe our construction of an airtight inebriometer that we have designed for this purpose. We also provide data that validates this apparatus and establishes a procedure for its use.

An airtight approach to the inebriometer: from construction to application with volatile anesthetics.

Drosophila melanogaster is an attractive model of familial Parkinson's disease, as flies with loss-of-function mutations of the parkin gene exhibit many pathologies observed in PD patients. Progressive motor deficits found in homozygous parkin mutants seem to result from mitochondrial pathology that causes indirect flight muscle and dopaminergic neuronal degeneration [1,2]. We have found that heterozygous parkin mutants have decreased lifespan, generally progressive motor dysfunction and olfactory deficits compared to control flies, suggesting that mutation of this gene produces a dominant phenotype. Tobacco smokers are dose-dependently less likely to develop PD [3,4]; subsequent in vitro and in vivo studies show that nicotine is protective in models of sporadic PD [6]. Literature addressing the potential protection by nicotine in Parkin loss-of-function models spans limited concentrations and selected time points in the organism's lifespan. We have found that parkin heterozygotes have late-onset climbing and flying deficits as well as decreased viability and olfactory deficits that precede motor defects. While chronic nicotine exposure decreases lifespan and climbing and flying abilities in control flies, it can improve viability and flying capability as well as rescue climbing and olfactory deficits in parkin heterozygotes. Dopaminergic neurons are spared in the parkin heterozygote, perhaps because this phenotype is less severe than in the homozygous parkin mutants. Nicotine pretreatment may be protective in sporadic PD patients and models; however, timely diagnosis remains to be an obstacle. Our results suggest that nicotine also may be protective in familial PD patients, who can be easily identified before motor symptoms occur.

Nicotine increases lifespan and rescues olfactory and motor deficits in a Drosophila model of Parkinson's disease.

Many biological response curves commonly assume a sigmoidal shape that can be approximated well by means of the 4-parameter nonlinear logistic equation, also called the Hill equation. However, estimation of the Hill equation parameters requires access to commercial software or the ability to write computer code. Here we present two user-friendly and freely available computer programs to fit the Hill equation - a Solver-based Microsoft Excel template and a stand-alone GUI-based "point and click" program, called HEPB.

Computational tools for fitting the Hill equation to dose-response curves.

Article	Year
Species differences in GnRH activation of the LHbeta promoter: role of Egr1 and Sp1. Molecular and cellular endocrinology\| PubMed ID: 12039067	2002
Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers. PLoS biology\| PubMed ID: 15719063	2005
An efficient genetic screen in Drosophila to identify nuclear-encoded genes with mitochondrial function. Genetics\| PubMed ID: 16849596	2006
Notch signaling controls germline stem cell niche formation in the Drosophila ovary. Development (Cambridge, England)\| PubMed ID: 17287246	2007
Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes. Genetics\| PubMed ID: 17720911	2007
Annual Drosophila research conference, 2008. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 18924240	2008
G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila. Nature methods\| PubMed ID: 19633663	2009
Shop talk: Annual Drosophila Research Conference, 2010. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 20865791	2010
Annual Drosophila research conference, 2011. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 21761487	2011
Annual Drosophila Research Conference, 2012. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 22592979	2012
An airtight approach to the inebriometer: from construction to application with volatile anesthetics. Fly\| PubMed ID: 23579237	2013
Nicotine increases lifespan and rescues olfactory and motor deficits in a Drosophila model of Parkinson's disease. Behavioural brain research\| PubMed ID: 23871228	2013
Computational tools for fitting the Hill equation to dose-response curves. Journal of pharmacological and toxicological methods\| PubMed ID: 25157754	2014

Gerald B. Call

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