Name: upright imaging of drosophila egg chambers
Uploaded: 2015-03-13T14:00:00.000+00:00
Duration: 12 min 29 s
Description: Watch this Scientific Journal Video about Upright Imaging of Drosophila Egg Chambers at JoVE.com

We appreciate assistance from members of the fly community, particularly Dr. Denise Montell, Dr. Lynn Cooley, and Dr. Pernille Rorth, for reagents. We thank Flybase, the Bloomington Drosophila Stock Center, and Developmental Studies Hybridoma Bank for information and providing fly stocks and antibodies, respectively. LM is supported by the Department of Education&#160;Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017) and by a NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036). A portion of the microscopy work was supported by NSF MRI grant DBI-0722569 and the Keith R. Porter Core Imaging Facility. Research was supported in part by a NSF CAREER Award (1054422) and a Basil O&#8217;Connor Starter Scholar Award from the March of Dimes, both awarded to MSG.

1. Dissecção da ovaríolos <ol><li> Transferir cerca de quinze 2-4 dias de idade moscas fêmeas e alguns machos a uma mosca frasco fresco alimentos com adição de levedura seca activa. Use algodão como um plug para os frascos, e adicione algumas gotas de água para o algodão para manter a umidade alta. </li><li> Coloque o frasco numa incubadora a 25 ° C durante 14-16 h, para maximizar o número de câmaras de 8-10 fase de ovo. NOTA: O tempo de incubação varia dependendo da temperatura e estágio desejado. </li><li> Prepare a mídia de dissecação, 0,1 M KPO 4 e NP40 soluções conforme necessário para o dia seguinte (ver Materiais). </li><li> Anestesiar as moscas usando CO 2, e colocar sob um microscópio de dissecção. Pipet várias gotas de mídia dissecção em cada cavidade no slide depressão vidro. </li><li> Segure fórceps em cada mão em um ângulo de aproximadamente 30 ° para o topo da tabela e orientar uma mosca fêmea com as asas para baixo. Com a mão não-dominante, pegar a fêmea com a pinça, agarrando-a no anterior do abdômen, perto do tórax, e mergulhe-a para a mídia dissecção na depressão de slides (Figura 1A inserção). </li><li> Com o outro par de fórceps, segure o exoesqueleto no ventral posterior do abdômen e perfurar. Agarre os ovários na extremidade posterior, em seguida, puxe-os para fora do abdômen e coloque no meio fresco, do outro lado do slide depressão. (Figura 1A inserção). Evite puxar para além do intestino. Descarte a carcaça. </li><li> Repita o procedimento com as outras moscas fêmeas até que todos os ovários são dissecados. Descartar os machos. </li><li> Com um par de fórceps, segure um ovário na extremidade posterior (perto das maiores câmaras de ovos), e com a outra mão use uma pinça para beliscar uma das câmaras de ovos mais anteriores (germário). (Figura 1A). </li><li> Lenta e progressivamente, puxe uma cadeia ovaríolo fora da bainha de ovário. Continue a dissecar ovarioles individualmente até que todas as câmaras de ovos desejados são removed. </li><li> Repita os passos de 1,7-1,8 para todos os ovários dissecados. </li><li> Uma vez completo, transferir ovarioles a um tubo de 0,6 ml usando uma pipeta de plástico. </li><li> Vamos ovarioles se depositam no fundo do tubo, em seguida, proceder à fixação e coloração etapas. </li></ol> 2. Immunofluorescent (IF) Coloração de ovaríolos <ol><li> Remova cuidadosamente media dissecção de ovarioles com uma pipeta. Cuidado para não remover quaisquer ovarioles ou câmaras de ovo. Deixe aproximadamente 50 l de ovarioles e meios de comunicação de dissecação. </li><li> Adicionar 50 mL de 16% de paraformaldeído para 150 mL de 0,1 M KPO 4 tampão em um tubo, e adicionar solução para câmaras de ovos para corrigi-los. Incubar, enquanto balançando para 10 min. CUIDADO: paraformaldeído é tóxico. </li><li> Remover fixador e lavar duas vezes com 0,5 ml de tampão de lavagem NP40. </li><li> Lavar duas vezes durante 15 minutos cada à temperatura ambiente. </li><li> Consulte o padrão se o protocolo 15 para as etapas subseqüentes. <ol><li> Resumidamente, após a fixação, adicionarOs anticorpos primários nas diluições apropriadas e incubar O / N a 4 ° C. Lavar em várias vezes NP40, em seguida, adicionar anticorpos secundários (1: 200) diluições. </li><li> Lavar várias vezes em NP40, em seguida, adicionar DAPI (1: 1000) durante 10 min, em seguida, repetir lavagens. Uma vez SE protocolo é completa, adicionar 200 ul de glicerol a 50% em PBS para as câmaras de ovos e deixar repousar à temperatura ambiente durante 2 horas. </li><li> Remova a solução, substitua-o por 70% de glicerol em PBS, e cubra com papel alumínio. Coloque amostra em 4 ᵒC até que esteja pronto para a montagem. Enquanto isso, continue com a etapa 3. </li></ol></li></ol> 3. Preparação Prévia de glicerina geléia Slides <ol><li> Medida pelo menos 4 ml de glicerina geléia com uma espátula e encher um tubo de cultura de células de vidro para a marca de meio caminho. Derreter glicerina geleia num banho de água a 55 ° C durante 30 min. </li><li> Verter uma pequena gota de glicerol, cerca de 300 ul, a partir da solução estoque em duas lâminas de microscópio. Utilizando um tecido, de glicerol em espalhar um la finayer através de cada um dos slides. Isto proporciona uma base e permite a fácil remoção de glicerina geleia no passo 5.6. </li><li> Corte a ponta fora de uma pipeta de ponta 1.000 ul filtrada. Use cut ponteira para transferir 1000-1500 ul de warm geléia glicerina lentamente para cada lâmina de microscópio. Tome cuidado para evitar bolhas. </li><li> Vamos lâminas glicerina geléia sentar por 5 min a RT para definir. </li><li> Coloque cada slide glicerina geléia em 60 mm x 15 milímetros placa de petri e cubra com filme plástico. Coloque a 4 ° CO / N. Opcionalmente, loja glicerina lâminas de geléia para até 5 dias a 4 ° C. </li></ol> 4. Montagem ovo Chambers <ol><li> Usando amostras do passo 2.5, pipeta várias gotas 3 ul de câmaras de ovos em 70% de glicerol através de uma glicerina geléia slide. Continue pipetagem 3 gotas ul até que todas as câmaras de ovos estão neste slide. Certifique-se de cada gota contém, pelo menos, dez câmaras de ovo. Enquanto isso, aqueça um tubo de cultura de glicerina geléia em um banho de água a 55 ° C. </li><li> Coloque o slide glicerina geléia com câmaras de ovo sob um microscópio de dissecção. Ajustar ampliação de modo a que apenas uma gota de câmaras de ovo está no campo de visão (figura 1B). </li><li> Usando uma agulha de calibre 30 como uma colher, pegar a câmara fase de ovo desejado. Quando necessárias, as câmaras de ovo desejados separados de uma cadeia ovaríolo utilizando a agulha, como uma faca. </li><li> Para evitar detritos que podem interferir com a imagem, a câmara de transferência de ovo para a segunda lâmina de glicerina geleia, com o anterior virada para a esquerda (Figura 1E -1). Repetir até que não haja, pelo menos, sete câmaras de ovo alinhados em uma coluna (Figura 1C). </li><li> Formar novas colunas de sete até que todas as câmaras de ovos desejados são transferidos para o segundo slide glicerina geléia. </li><li> Rasgue um pequeno pedaço de tecido e torção. Use-o para absorver o excesso de PBS-glicerol das câmaras de ovo levemente tocando cada um. Tome cuidado para não remover câmaras de ovo. </li><li> Corte oponta de uma pipeta de ponta de 200 ul filtrada. Utilize este para transferir 150 mL de glicerina geléia para cobrir todas as colunas de câmaras de ovo. NOTA: A quantidade de geléia glicerina necessário para cobrir as colunas é baseado em etapas da câmara de ovos e número de colunas. Este valor pode ser ajustado. </li><li> Deixe câmaras de ovos montados sentar-se durante 5 min à temperatura ambiente até que a camada superior de glicerina geleia é completamente solidificado (Figura 1D). </li><li> Colocar a lâmina de câmaras de ovos montados em 60 mm × 15 milímetros placa de petri e cubra com filme plástico. Armazenar a 4 ° CO / N para permitir que as camadas glicerina geléia para solidificar juntos (lugar no escuro, se há uma preocupação com a fotodegradação). </li></ol> 5. Preparação de ovo Chambers for Imaging <ol><li> Coloque slides das câmaras de ovos montados sob um microscópio de dissecção. Ajustar ampliação de modo a que apenas uma coluna de câmaras de ovo está no campo de visão. </li><li> Usando um ângulo de 45 ° bisturi miniatura, corte um straight linha ao longo do lado direito da primeira coluna de câmaras de ovo (próxima da face posterior das câmaras de ovo). (Figura 1D-E) </li><li> Em seguida, cortado por cima da primeira câmara de ovo no topo e por baixo da última câmara de ovo na coluna. Neste ponto, a coluna de câmaras de ovo deve ser cortada em três lados. </li><li> Usando um microlanceta, corte ao longo do lado esquerdo da coluna, tão perto quanto possível do lado anterior das câmaras de ovo (Figura 1E) -3. </li><li> Pipetar 100 mL de frio 1X PBT sobre a coluna corte. </li><li> Use uma espátula rodada gumes para remover o excesso de geléia glicerina em torno de três lados da coluna corte de câmaras de ovo e descartar, deixando o resto no slide. </li><li> Insira a espátula no corte feito na face anterior das câmaras de ovos e separar a coluna do bloco de glicerina geléia primária. </li><li> Preparar amostras em posição invertida (5.8.1) ou vertical (5.8.2) de microscopia para qualquer um. <ol><li> Para microscópio invertido: t Transferênciaele coluna de câmaras de ovos para uma lamela com face anterior das câmaras de ovo virado para baixo. Repita os passos 5,2-5,8 até que todas as colunas de glicerina geleia foram removidos e montados. Adicione um par de gotas de 50% PBS-glicerol para cada bloco de câmaras de ovos para evitar que sequem. Câmaras de ovos imagem diretamente sobre lamela. </li><li> Para microscópio vertical: Transferir a coluna de câmaras de ovo para uma lâmina de microscópio com o lado anterior para cima. Repita os passos 5,2-5,9 até que todas as colunas de glicerina geleia foram removidas e montadas em lâminas de microscópio. Adicione um par de gotas de 50% PBS-glicerol para cada bloco de câmaras de ovo e blocos de cobertura com uma lamela ½ # 1. </li></ol></li></ol>

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The authors have no competing financial interests.

Aqui nós descrevemos um método para montar e pequena imagem, o desenvolvimento de câmaras de ovos de uma perspectiva end-on. Técnicas comuns para câmaras de ovos de imagem são otimizados para vistas laterais e permitir principalmente a visualização precisa das células do folículo medio-lateral quando coradas com anticorpos fluorescentes. O uso de Z-stacks ou 3-D reconstruções auxilia na visualização de vários planos focais, mas ainda é insuficiente para a resolução de sub-celular dos pólos de câmaras...

Estudo da Drosophila melanogaster tem proporcionado grandes insights sobre a regulação genética de uma ampla gama de fenômenos. Em particular, tem havido uma extensa pesquisa sobre o desenvolvimento do ovo, porque oogenesis fornece uma maneira dócil para investigar vários processos de desenvolvimento diferentes, incluindo padrões de tecidos, alterações de polaridade celular, comutação do ciclo celular, e regulação de translação 1,2,3,4. Um evento morphogenic importante durante oogenesis é a especificação, aquisição de motilidade, e migração de um conjunto de células, chamadas de células de fronteira (revisto em 5). Uma vez que a migração celular é uma característica fundamental da morfogénese dos animais, e porque a regulação genética do presente processo é bem conservada, determinados mecanismos de moscas são susceptíveis de ser importante em outros contextos. Assim, estamos investigando o controle molecular da migração celular fronteira. Para os nossos estudos, desenvolvemos um novo método para observar os pólos anterior de desenvolvimento de ovos,onde surgem as células de fronteira, para examinar como elas se desenvolvem em detalhe. Dentro do ovário, câmaras de ovo em várias fases de desenvolvimento existir ao longo das cadeias chamadas ovarioles, que são envolvidas por uma bainha fina (Figura 1A). Cada câmara ovo vai continuar a formar um ovo. Durante oogenesis, vários tipos de células diferentes deve desenvolver-se de forma coordenada. O D. câmara melanogaster ovo é composto por 16 células germinativas, incluindo um oócito, cercado por uma única camada de epitélio folicular 6. Um pequeno número de células especializadas, chamadas células polares, surgem as anteriores e posteriores pólos do epitélio. As células de fronteira 6-8 originam no epitélio anterior da câmara de ovo, induzida pelas células polares 5,7. Em meados de oogénese (etapa 9), as células de fronteira desanexar das suas vizinhas e migram entre as células de enfermagem para atingir o oócito na parte posterior da câmara do ovo 5,7. Este movimento deve ser realizadoenquanto que as células permanecem na fronteira um cluster circundante duas células polares não-móveis, tornando este um tipo de migração celular colectiva. Migração bem-sucedida do grupo de células de fronteira garante o bom desenvolvimento da micrópila da casca do ovo, que é necessário para a fertilização. As células polares anteriores instruir destino celular fronteira ativando uma cascata de transdução de sinal. Células polares secretar uma citocina, Unpaired (UPD), que se liga a um receptor de membrana, Domeless (DOME), na vizinha células foliculares durante a fase de desenvolvimento do oócito 8 8,9. A ligação da UPD provoca Janus tirosina quinase (JAK) para fosforilar o transdutor de sinal e ativador de transcrição (STAT) 8,10,11. STAT, em seguida, move-se para o núcleo para activar a transcrição. Células de Fronteira lenta (SLBO) é um fator de transcrição que é um alvo direto da transcrição do STAT e também é necessário para a migração celular fronteira 12. Vistas laterais de câmaras de ovos indicam tatividade STAT chapéu é regulada em um gradiente através do epitélio anterior 8,11,13. Células do folículo mais próximos às células polares têm os mais altos níveis de STAT ativado, assim, tornam-se células de fronteira e invadir o tecido germinal adjacente. Para entender como as células de fronteira são especificados dentro e destacar do epitélio, precisamos observar como o tecido é organizado. Se virmos câmaras de ovos de uma perspectiva anterior-on, esperaríamos simetria radial da atividade STAT nas células foliculares que cercam as células polares. Um fim-em vista também mostram mais precisamente as diferenças de proteínas de membrana e interfaces célula-célula antes e durante o descolamento do que comparando células em diferentes planos focais. Porque câmaras de ovo são oblongas e ligados uns aos outros por células pedunculares, estabelecem-se em lâminas lateralmente, tornando-o difícil de observar a arquitectura anterior. Assim, muita informação sobre as células nos pólos da câmara de ovo temsido inferida a partir de vistas laterais. Embora algumas informações podem ser obtidas através de algoritmos reconstruções 3-D de secções ópticas, espalhamento de luz, fotodegradativas e limites mais pobres da resolução no eixo Z tornar essa informação menos detalhada e confiável na ausência de técnicas caras, como microscopia de super-resolução 14 . Outros tipos de imagem baseado em seção (por exemplo, microscopia eletrônica ou microtome seccionamento) exigem extensa manipulação de tecidos, incluindo a desidratação, aumentando a probabilidade de artefatos. Assim, foi desenvolvido um novo método para a imagem D. câmaras melanogaster ovos, enquanto na posição vertical. Este método já provou útil para elucidar como células móveis estão fadados (ver resultados representativos e Manning et al, em revisão), e é provável que seja mais amplamente útil em estudos de outros aspectos da oogenesis.

<table><tbody><tr><td>0.1 M Potassium phosphate Buffer (KPO&lt;sub&gt;4&lt;/sub&gt; Buffer)</td><td></td><td></td><td>Add 3.1 g of NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;H&lt;sub&gt;2&lt;/sub&gt;O and 10.9 g of Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (anhydrous) to distilled H&lt;sub&gt;2&lt;/sub&gt;O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 month at 4&amp;deg;C.</td></tr><tr><td>16% Paraformaldehyde aqueous solution, EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>methanol-free to preserve GFP fluorescence</td></tr><tr><td>30G x 1/2 inch needle&amp;nbsp;</td><td>VWR</td><td>BD305106</td><td>Regular bevel</td></tr><tr><td>Active Dry Yeast</td><td>Genesee Scientific</td><td>62-103</td><td>To fatten female flies</td></tr><tr><td>Bovine Serum Albumin</td><td>PAA-cell culture company</td><td>A15-701</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11295-10</td><td>Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td></tr><tr><td>DAPI (4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride)</td><td>Sigma Aldrich</td><td>D9542</td><td>5 mg/ml stock solution&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16140-071</td><td>Added to supplement dissection medium (10%)&amp;nbsp;</td></tr><tr><td>Glass culture tube</td><td>VWR</td><td>47729-576</td><td>14 ml</td></tr><tr><td>Glass Depression Slides</td><td>VWR</td><td>470019-020</td><td>1.2 mm Thick, Double Cavity</td></tr><tr><td>Glycerin Jelly&amp;nbsp;</td><td>Electron Microsocpy Sciences</td><td>17998-10</td><td>Mounting media</td></tr><tr><td>Glycerol</td><td>IBI Scientific</td><td>IB15760</td><td>70% in PBS</td></tr><tr><td>IGEPAL CA-630</td><td>Sigma Aldrich</td><td>I3021-500ML</td><td>(interchangable for Nonidet P-40)&amp;nbsp; Used in NP40 Wash Buffer&amp;nbsp;</td></tr><tr><td>Leica Fluorescent Stereoscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leica SP5 Confocal Microscope</td><td>Leica Microsystems</td><td></td><td>40x/0.55NA dry objective&amp;nbsp;</td></tr><tr><td>Micro spatula&amp;nbsp;</td><td>VWR</td><td>82027-518</td><td>Stainless steel</td></tr><tr><td>Microscope Slide</td><td>VWR</td><td>16004-368</td><td>75x25x1 mm</td></tr><tr><td>NP40 Wash Buffer</td><td></td><td></td><td>50 mM TRIS-HCl, 150 mM NaCl, 0.5% Ipegal, 1 mg/ml BSA, and 0.02% sodium azide</td></tr><tr><td>Penicillin-Streptomycin-Glutamine</td><td>Life Technologies</td><td>10378-016</td><td>Added to supplement dissection medium (0.6X)</td></tr><tr><td>Petridish- Polysterine, sterile</td><td>VWR</td><td>82050-548</td><td>60 W x 15 H mm</td></tr><tr><td>Phosphate Buffer Saline</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td>10 packs of Powder</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma Aldrich</td><td>P3786-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P9791-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Schneider&amp;rsquo;s Insect Medium</td><td>Life Technologies</td><td>&lt;br/&gt; 11720018</td><td>With L-glutamine and sodium bicarbonate</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>S2002-25G</td><td>Used in fluorescent antibody staining</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S3014-500G</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>TRIS-HCl</td><td>IBI Scientific</td><td>IB70144</td><td>1 M TRIS-HCl, pH 7.4</td></tr><tr><td>Volocity 3D Image Analysis Software</td><td>PerkinElmer</td><td></td><td>For processing confocal Z-stacks</td></tr></tbody></table>

upright imaging of drosophila egg chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

O método de imagem na vertical nos permitiu ver diretamente a organização das células do epitélio folicular anterior na fase 8. Um marcador geral para o destino células do folículo, o Absent Eyes (EJA) de proteínas, bem como o marcador de DNA nuclear DAPI, mostrou ainda expressão através deste campo de células, e demonstrou que todas as células podem ser vistos com intensidades de coloração semelhante (Figura 2B &quot;). As proteínas reguladas, em resposta à UPD citoquina, no entanto, mo...

Watch this Scientific Journal Video about Upright Imaging of Drosophila Egg Chambers at JoVE.com

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Imagem Vertical de<em&gt; Drosophila</em&gt; Câmaras de ovo

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

upright-imaging-of-drosophila-egg-chambers

Research

JoVE Journal

Developmental Biology

Upright Imaging of Drosophila Egg Chambers

7.6K visualizações.  University of Maryland Baltimore County. The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Vídeo: Upright Imaging of Drosophila Egg Chambers

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Vivo-imaging da<em&gt; Drosophila</em&gt; Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Biologia do Desenvolvimento

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

Imagens de cálcio na<em&gt; Drosophila</em&gt; At Activation Egg

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mesoscópica de fluorescência para Tomografia In-vivo Imaging de desenvolvimento Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Biologia

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Imagem e Análise da Dinâmica de Orientação e Crescimento tecidual na Epitélia Drosophila em Desenvolvimento durante os estágios pupal

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

Imagem Animal inteira de Drosophila melanogaster usando tomografia microcomputada

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

Visualizando os efeitos do dano oxidativo nas câmaras de ovos de Drosophila usando imagens ao vivo

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

Migração Nuclear no Oócito de Drosophila

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imagem vertical de Drosophila Os embriões

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Detecção de Anastasis na Vivo por CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Medicina

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

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