Patrick Cafferty é apoiado por uma bolsa de pós-doutorado da Sociedade de Esclerose Múltipla do Canadá.

Parte 1: Pré-experimental set-up. <ol><li> Uma semana de antecedência, mate moscas para gerar larva que expressam GFP sob o controle de um promotor glial específico. Para nosso experimento foi visualizada GFP marcado com uma seqüência de localização nuclear expresso em células gliais usando a polaridade invertida (repo) promotor (3, 4). </li><li> Prepare tampa desliza pelo menos um dia de antecedência por imersão 18 milímetros lamínulas redondas de 10 minutos em 1% de poli-L-lisina solução e ar seco durante a noite. </li><li> No dia antes do experimento, limpar uma câmara de cultura Chamlide magnética por imersão componentes durante a noite em etanol 70%. </li><li> No dia do experimento, preparar meios de cultura pela adição de soro fetal bovino, penicilina-estreptomicina solução, e de insulina para 10 ml de meio de insetos Schneider, utilizando técnica asséptica. As concentrações de trabalho são: 1X de soro fetal bovino; 100U/ml penicilina; estreptomicina 0.1mg/ml; insulina e 0,2 mg / ml em meio de insetos Schneider (modificado de (5)). </li><li> Permitir que a câmara de cultura para ar seco em uma capa de cultura de células. Lavar o etanol residual da câmara de cultura por lavagem com meio de cultura preparado. </li></ol> Parte 2: Dissecção do complexo Drosophila olho-cérebro. <ol><li> Selecione uma larva do terceiro ínstar vagando do lado de um frasco de voar e coloque em uma gota de meio de cultura refrigeradas em uma placa de Petri sobre gelo por alguns minutos. Refrigeração da larva é recomendada para diminuir as contrações peristálticas do corpo para facilitar a dissecção. </li><li> Coloque a larva refrigerados em uma gota de meio de cultura em um prato Sylgard revestido sob um microscópio de dissecação. </li><li> Sob o microscópio de dissecção, use um par de fórceps Dumont multa para agarrar firme a larva aproximadamente um terço do caminho entre a extremidade posterior. Aguarde até que a larva extrudes ganchos sua boca a partir do final anterior. Segure a boca ganchos com um segundo par de fórceps sobre extensão completa. Para dissecar a larva, puxe lentamente os dois pares de fórceps em direções opostas. O complexo olho-cérebro, junto com as glândulas salivares, órgãos, gordura e tecido imaginal, vai puxar para longe do corpo larval. Não puxe a boca ganchos muito rapidamente caso contrário eles vão arrancar do complexo olho-cérebro. </li><li> Apare as glândulas salivares, corpos gordos, e os discos imaginal usando uma tesoura ultra-fino clipper. Os dois hemisférios do cérebro e olho-discos serão anexados ao cordão nervoso ventral e ganchos boca. </li><li> Coloque uma lamínula 18 milímetros em uma lâmina de vidro. Deixe uma borda da lamínula pendurado na borda do slide para depois pegar a lamínula. Adicionar uma gota de meio de cultura para a lamínula 18 mm. Transferência do complexo olho-cérebro no meio de cultura, segurando os ganchos na boca com uma pinça. Não entender o complexo olho-cérebro diretamente com a pinça ou o tecido será danificado. </li><li> Com uma tesoura de dissecção fina, corte na boca ganchos do complexo olho-cérebro e descartar. Remoção dos ganchos boca é importante para geração de imagens ao vivo como os ganchos na boca vai continuar a contrair-se o meio de cultura fazendo com que o tecido se mova durante microscopia. </li><li> Para visualizar a migração glial no disco imaginal olho, corte cuidadosamente a haste óptica, o tecido fino que liga o cérebro eo disco olho e afasta ou descartar o cérebro (ver figura 1A). Para visualizar glia dentro do talo óptico, deixe o cérebro, talo óptico e disco imaginal olho intacto (ver figura 1E). </li><li> Pick up a lamela com a pinça e desenhar um círculo em volta do tecido embaixo de interesse com um marcador permanente. Este círculo vai ajudar na localização do tecido para microscopia na Parte 4. </li></ol> Parte 3: Montagem do disco imaginal olho em uma câmara de cultura magnético. <ol><li> Utilizando uma pinça, segure a ponta pendurada da lamela. Transferir a lamela ao tabuleiro inferior da câmara de Chamlide magnética sem perturbar o tecido em cultura. </li><li> Coloque o silicone O-ring para o corpo principal da câmara Chamlide. Instale o corpo principal no topo da placa inferior. </li><li> Lentamente, adicionar meio de cultura para a câmara. Não adicione o meio de cultura muito rapidamente ou o tecido de interesse será perturbado. Não encher completamente a câmara para permitir a troca gasosa sob a tampa. </li><li> Delicadamente, coloque a tampa invisível no topo da câmara Chamlide. </li></ol> Parte 4: Visualização de migração de células gliais no disco olho. <ol><li> Coloque a câmara de cultura no palco de um microscópio confocal ou fluorescência. Localize a amostra usando o círculo na lamela como uma referência na baixa ampliação. </li><li> Foco na amostra usando uma lente de 40x. Permitir que o tecido para resolver sobre a lamínula. Capturar imagens do disco óptico do olho ou perseguir a cada 10-15min mais de 3-4 horas. O tecido pode contorcer-se e mover-se fora de foco. Manualmente focando o tecido antes da captura de imagem irá aliviar ªé problema. </li></ol> Parte 5: Os resultados representativos: Realizado corretamente, o nosso protocolo permitiu coletar uma série de imagens de GFP-tagged células gliais que migraram do talo no disco óptico do olho imaginal (Figura 1 BD). Enquanto imagens ao vivo por um período de 60 minutos foi suficiente para observar as mudanças nas posições dos núcleos gliais dentro de um disco de tipo selvagem olho imaginal, núcleos gliais em um mutante de um gene necessário para a migração de células gliais falhou completamente para sair o talo óptica (setas Figura 1 FH). Temos cultivadas olho-cérebro complexos para períodos de até 240 minutos antes de observar a deterioração do tecido cultivado. Após os 240 minutos de tempo-ponto, começamos a observar as células GFP positivas no meio de cultura em torno cultivadas olho-cérebro complexos (seta Figura 2 C). Em GFP disso irá acumular em pontos difusa em todos os tecidos, sugerindo uma quebra na integridade do tecido. <img src="/files/ftp_upload/1155/figure1.png" alt="" width="589" height="241" /> Figura 1: imagem ao vivo de GFP-tagged núcleos gliais do tipo selvagem e mutante desenvolvimento sistemas visual. A, E) Imagens tiradas com contraste de interferência diferencial (DIC) de microscopia do tipo selvagem e mutante cultivadas olho-imaginal discos (ED). No tipo selvagem, células gliais nascem e migram da haste óptica (OS) no olho do disco. O cérebro foi removido para facilitar o achatamento e imagem do disco imaginal olho. BD) microscopia de fluorescência do selvagem disco olho tipo cultivado no imaginal (A) em 0, 30 e 60 pontos tempo minucioso revela GFP-tagged núcleos gliais migrar dentro do disco olho. FH) microscopia de fluorescência do complexo olho-cérebro mutante em (E) em 0, 30 e 60 pontos tempo minucioso demonstra uma estagnação da GFP-tagged núcleos gliais dentro do talo óptico (seta). O cérebro (BR) foi deixado ligado à haste óptica para facilitar a criação de imagens de glia dentro do caule. <img src="/files/ftp_upload/1155/Figure_2.png" alt="" width="600" height="156" /> Figura 2: imagens ao vivo de GFP-tagged membranas gliais em um disco de tipo selvagem olho imaginal. Conseguimos cultivadas olho-cérebro complexos para 240 minutos períodos. Tecidos cultivados mais de 240 minutos começa a quebrar. O meio de cultura em torno do disco de olho imaginal (ED), perseguir óptica (OS), e lobo cerebral (BR) a 0 (A) e 150 minutos (B), em comparação com o 300 minutos de tempo-ponto (C), é livre da GFP células positivas, indicado por uma seta.

<ol>
	<li>Aigouy, B., Lepelletier, L., Giangrande, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+chain+migration+requires+pioneer+cells.">Glial chain migration requires pioneer cells.</a> J. Neurosci. 28, 11635-11641 (2008).</li><li>Silies, M., Yuva, Y., Engelen, D., Aho, A., Stork, T., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+migration+in+the+eye+disc.">Glial cell migration in the eye disc.</a> J. Neurosci. 27, 13130-13139 (2007).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sepp, K. J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+lacZ+enhancer+trap+lines+to+GAL4+lines+using+targeted+transposition+in+Drosophila+melanogaster.">Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.</a> Genetics. 151, 1093-1101 (1999).</li><li>Gibson, M. C., Patel, A. B., Nagpal, R., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+geometric+order+in+proliferating+metazoan+epitheia.">The emergence of geometric order in proliferating metazoan epitheia.</a> Nature. 442, 1038-1041 (2006).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends Neurosci. 24, 251-254 (2001).</li></ol>

The authors have nothing to disclose.

Neste protocolo, descrevemos a observação da migração de células gliais no disco imaginal olho através de microscopia ao vivo. No nosso exemplo de tipo selvagem (Figura 1 AD), foi utilizado um marcador GFP nuclear para observar a movimentação das células gliais no disco do olho ao longo de uma hora. Em um mutante de um gene candidato necessários para a migração de células gliais actualmente em estudo no nosso laboratório, observamos um adiamento de núcleos de células gliais no talo óptico duran...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Poly-L-lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P8920</td>
<td>&nbsp;</td>
<tr>
<td>Schneider's Insect Media</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P4458</td>
<td>&nbsp;</td>
<tr>
<td>Insulin solution from bovine pancreas</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I0516</td>
<td>&nbsp;</td>
<tr>
<td>Chamlide Magnetic chamber</td>
<td>Tool</td>
<td>Live cell Instrument</td>
<td>CM-R-10</td>
<td>35 mm dish type chamber for 18 mm coverslip</td>
</tr>
<tr>
<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>11251-20</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>Any fluorescent imaging system that has the necessary filters and excitation for GFP can be used. </td>
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live imaging of glial cell migration in the drosophila eye imaginal disc

Células gliais de ambos os vertebrados e organismos invertebrados devem migrar para regiões de destino final para ensheath e apoio neurônios associados. Embora o progresso recente tem sido feito para descrever a migração de células gliais na ala desenvolvimento pupal (1), estudos de<em&gt; Drosophila</em&gt; Migração de células gliais têm tipicamente envolvidos no exame de tecido fixo. Análise microscópica de células móveis ao vivo oferece a capacidade de examinar o comportamento celular em todo o processo migratório, incluindo a determinação da taxa de e mudanças na direção do crescimento. Emparelhado com o uso de ferramentas genéticas, imagens ao vivo pode ser usado para determinar os papéis mais precisos para genes específicos no processo de desenvolvimento. Os trabalhos anteriores por Silies<em&gt; Et al.</em&gt; (2) descreveu a migração de células gliais provenientes do talo óptico, uma estrutura que liga o olho eo cérebro em desenvolvimento, no disco imaginal olho no tecido fixo. Aqui destacamos um protocolo para a análise da migração de células gliais na<em&gt; Drosophila</em&gt; Disco olho imaginal. Aproveitamos uma linha de Drosophila que expressa GFP em células gliais em desenvolvimento para acompanhar a progressão de células gliais no tipo selvagem e mutante em animais.

Watch this Scientific Journal Video about Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc at JoVE.com

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Aqui nós descrevemos um protocolo para analisar a migração de células gliais no olho Drosophila desenvolvimento, utilizando análise microscópica ao vivo emparelhado com GFP tag células gliais.

Imagens ao vivo da migração das células da glia no Disco Drosophila Eye Imaginal

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

live-imaging-glial-cell-migration-drosophila-eye-imaginal

Research

JoVE Journal

Biology

10.6K visualizações.  University of British Columbia - UBC. Aqui nós descrevemos um protocolo para analisar a migração de células gliais no olho Drosophila desenvolvimento, utilizando análise microscópica ao vivo emparelhado com GFP tag células gliais.

Vídeo: Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Dissecação de discos imaginais de 3 larvas Drosophila Instar

Biologia

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Visualizando o Live Drosophila Junção neuromuscular-Glial com corantes fluorescentes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

Elaboração de Drosophila Os embriões para Live-Imaging Usando o protocolo gota suspensa

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

A Novel Reporter RFP para ajudar na visualização do Disco Imaginal Eye em Drosophila</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Imagens ao vivo de Drosophila melanogaster Embrionárias Migrações hemócitos

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imagem Alterar Forma Celular em Viver Drosophila Os embriões

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

Imagens ao vivo de Proteínas GFP-rotulados em<em&gt; Drosophila</em&gt; Oócitos

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

O tomate / GFP-FLP / FRT Method for Imaging ao vivo de Mosaico Adulto<em&gt; Drosophila</em&gt; As células fotorreceptoras

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Dissection and Mounting of Drosophila 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 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Dissecção e montagem de<em&gt; Drosophila</em&gt; pupais Discos Eye

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