Frequent Unanticipated Alleles of <i>lethal giant larvae</i> in Drosophila Second Chromosome Stocks

Roegiers, Fabrice; Kavaler, Joshua; Tolwinski, Nicholas S.; Chou, Yu-Ting; Duan, Hong; Bejarano, Fernando; Zitserman, Diana; Lai, Eric C.

doi:10.1534/genetics.109.101808

Cited by 28 publications

(30 citation statements)

References 28 publications

(38 reference statements)

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“…Among these are the Polycomb group (PcG) genes, which in some studies have been proposed to act as dominant suppressors of TPE [Su(TPE)] and whose encoded proteins have been found to be located at the telomeric regions of polytene chromosomes (6). These findings indicate that TPE is a distinctive class of chromatin silencing which shares mechanistic features with both pericentromeric heterochromatin and PcG-mediated silencing of developmental regulators.The extent to which PcG proteins and other reported Su(TPE) bind at TAS repeats and modify TPE, though, has become less clear since the finding of a high incidence of TPE-suppressing terminal deletions on chromosome 2L in public Drosophila mutant stocks (38,46). This leads to a high rate of false-positive identifications of Su(TPE), in which the modifying activity is attributable to the 2L deletion, which eliminates the TAS repeats at that location and suppresses TPE in trans, rather than to the mutant gene being tested (38).…”

mentioning

confidence: 99%

Protein Landscape atDrosophila melanogasterTelomere-Associated Sequence Repeats

Antao

Mason

Déjardin

et al. 2012

Molecular and Cellular Biology

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eThe specific set of proteins bound at each genomic locus contributes decisively to regulatory processes and to the identity of a cell. Understanding of the function of a particular locus requires the knowledge of what factors interact with that locus and how the protein composition changes in different cell types or during the response to internal and external signals. Proteomic analysis of isolated chromatin segments (PICh) was developed as a tool to target, purify, and identify proteins associated with a defined locus and was shown to allow the purification of human telomeric chromatin. Here we have developed this method to identify proteins that interact with the Drosophila telomere-associated sequence (TAS) repeats. Several of the purified factors were validated as novel TAS-bound proteins by chromatin immunoprecipitation, and the Brahma complex was confirmed as a dominant modifier of telomeric position effect through the use of a genetic test. These results offer information on the efficacy of applying the PICh protocol to loci with sequence more complex than that found at human telomeres and identify proteins that bind to the TAS repeats, which might contribute to TAS biology and chromatin silencing. P roteomic analysis of isolated chromatin segments (PICh) was developed as an unbiased method of identifying proteins that physically interact with a specific locus in the genome (11), and this was done by using the telomeres of mammalian cells as a target. Telomeres are found in multiple copies of a simple repeat sequence and so do not offer the same challenge to the use of PICh as other genomic loci do. In this work, we applied PICh to the telomere-associated sequence (TAS) repeats of Drosophila to demonstrate the efficacy of the technology and to learn about the biology of these repeats.TAS repeats are found in the subtelomeric region of chromosomes 2, 3, and X and nucleate a particular kind of heterochromatin which is responsible for the telomeric position effect (TPE; for a review, see reference 37). As also seen with pericentromeric heterochromatin-mediated position effect variegation (PEV), reporter genes inserted at the TAS repeats, or between the TAS repeats and the telomeric retrotransposon (HTT) arrays, variegate. The extent of gene silencing depends on the size, and hence the strength of the transcription activating effect, of the HTT array and the transcription-repressing activity of the TAS repeats (28). Interestingly, though, most modifiers of PEV [Mod(PEV)] have no effect on TPE; in fact, very few modifiers of TPE [Mod(TPE)] have been unambiguously described so far. Among these are the Polycomb group (PcG) genes, which in some studies have been proposed to act as dominant suppressors of TPE [Su(TPE)] and whose encoded proteins have been found to be located at the telomeric regions of polytene chromosomes (6). These findings indicate that TPE is a distinctive class of chromatin silencing which shares mechanistic features with both pericentromeric heterochromatin and PcG-mediated silencing of de...

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mentioning

confidence: 99%

Protein Landscape atDrosophila melanogasterTelomere-Associated Sequence Repeats

Antao

Mason

Déjardin

et al. 2012

Molecular and Cellular Biology

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“…3). Using the muscle-specific 24b-Gal4 driver (Brand and Perrimon, 1993) to express UAS-lgl (Roegiers et al, 2009) in the postsynaptic muscle cells of otherwise lgl null mutants, we observed improved larval motility and growth rates similar to heterozygous siblings, even when reared in competition (data not shown). At the NMJ, however, postsynaptic LGL expression provided no improvement of synaptic morphology (Fig.…”

Section: Resultsmentioning

confidence: 89%

“…RNAi experiments were performed using a w; lglRNAi/lgl-RNAi line (w; P{GD4047}v51247; Dickson et al, 2007) crossed to either the muscle-specific Gal4 driver 24b (w; 24b-Gal4/24b-Gal4; Brand and Perrimon, 1993;Nitabach et al, 2006;Rohrbough and Broadie, 2010) or the neuron-specific Gal4-driver elav (w; elav-Gal4/y; Luo et al, 1994;Rohrbough and Broadie, 2010). Rescue experiments were done on lines generated from a cross between UAS-lgl; lgl u334 /CyOKrGFP males (Roegiers et al, 2009), and virgin females from (1) lgl 1 /croKrGFP; uh1-Gal4 for ubiquitous expression (w; +; uh1-Gal4; Wodarz et al, 1995), (2) lgl 1 /CyOKrGFP; 24b-Gal4 for muscle expression or (3) elav-Gal4; lgl 1 /CyOKrGFP for neuronal expression. To control for background effects of Gal4 drivers, a single copy of each Gal4 driver alone was crossed into the lgl control background (uh1-Gal4/+, 24b-Gal4/+ and elav-Gal4/+).…”

Section: Genetic Stocksmentioning

confidence: 99%

The cell polarity scaffold lethal giant larvae regulates synapse morphology and function

Staples

Broadie

2013

Journal of Cell Science

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SummaryLethal Giant Larvae (LGL) is a cytosolic cell polarity scaffold whose loss dominantly enhances neuromuscular junction (NMJ) synaptic overgrowth caused by loss of the Fragile X Mental Retardation Protein (FMRP). However, direct roles for LGL in NMJ morphological and functional development have not before been tested. Here, we use confocal imaging and two-electrode voltage-clamp electrophysiology at the Drosophila larval NMJ to define the synaptic requirements of LGL. We find that LGL is expressed both preand postsynaptically, where the scaffold localizes at the membrane on both sides of the synaptic interface. We show that LGL has a cell autonomous presynaptic role facilitating NMJ terminal branching and synaptic bouton formation. Moreover, loss of both pre-and postsynaptic LGL strongly decreases evoked neurotransmission strength, whereas the frequency and amplitude of spontaneous synaptic vesicle fusion events is increased. Cell-targeted RNAi and rescue reveals separable pre-and postsynaptic LGL roles mediating neurotransmission. We show that presynaptic LGL facilitates the assembly of active zone vesicle fusion sites, and that neuronally targeted rescue of LGL is sufficient to ameliorate increased synaptic vesicle cycling imaged with FM1-43 dye labeling. Postsynaptically, we show that loss of LGL results in a net increase in total glutamate receptor (GluR) expression, associated with the selective elevation of GluRIIB subunit-containing receptors. Taken together, these data indicate that the presynaptic LGL scaffold facilitates the assembly of active zone fusion sites to regulate synaptic vesicle cycling, and that the postsynaptic LGL scaffold modulates glutamate receptor composition and function.

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“…These images resemble scanning electron micrographs of fly cuticle, but can be acquired from live animals, and allow us to simultaneously visualize cuticular morphology and expression of GFP fluorescence 6,7,12 .…”

Section: Discussionmentioning

confidence: 99%

Live-cell Imaging of Sensory Organ Precursor Cells in Intact <em>Drosophila</em> Pupae

Zitserman¹,

Roegiers²

2011

JoVE

Self Cite

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Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissuespecific Gal4 drivers and GFP-tagged fusion proteins 1,2 .This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8 ). Video LinkThe video component of this article can be found at https://www.jove.com/video/2706/ Protocol Required Materials: Dissection stereo-microscope, Double-sided tape, standard microscope slide and coverslip, dissection forceps (size 5 or 5.5), soft-bristled brush, silicone vacuum grease, 5cc syringe, Whatman paper, confocal or epifluorescence microscope with digital camera and image acquisition software. Pupal Dissection1. Set up a cross (using the appropriate combination of Gal4 line and a GFP tagged fusion protein under UAS control) or place flies from a stock you wish to image in several fresh vials at 25°C. 2. SOP cells generally begin to proliferate on the pupal thorax at eighteen hours after the onset of pupariation, we therefore select "white" pupae from the appropriate fly stock or cross. White pupae have the pupal morphology, but have an unpigmented pupal case,...

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Frequent Unanticipated Alleles of lethal giant larvae in Drosophila Second Chromosome Stocks

Cited by 28 publications

References 28 publications

Protein Landscape atDrosophila melanogasterTelomere-Associated Sequence Repeats

Protein Landscape atDrosophila melanogasterTelomere-Associated Sequence Repeats

The cell polarity scaffold lethal giant larvae regulates synapse morphology and function

Live-cell Imaging of Sensory Organ Precursor Cells in Intact <em>Drosophila</em> Pupae

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