We thank the Bloomington Stock Center (Bloomington, USA), Dirk Bohmann, Katja Br&#252;ckner and Istvan Ando for fly stocks, and antibodies. We thank Marek Jindra and Colin Donohoe for comments on the manuscript. This work was supported by the Sofja Kovalevskaja Award to M.U. from the Alexander von Humboldt Foundation and DFG project UH243/1-1 to M.U. from the German Research Foundation.

NOTA: Este trabalho utiliza o eyFLP1; act&gt; y +&gt; Gal4, UAS-GFP; Linha tester FRT82B, banheira-Gal80 marcm 82B Verde 11. Cruzando as virgens testador marcm 82B Verdes para machos de estirpes tais como w; UAS-um; UAS-b RNAi, FRT82B c mut genótipo irá produzir descendência em que a recombinação mitótico irá ocorrer entre os braços direitos dos cromossomos homólogos 3º. Desta forma, os clones mutante homozigótica para o gene c localizada distalmente ao local FRT82B irá ser gerado dentro do neuroepitélio EAD e cérebro. Estes clones irá expressar GFP, e um transgene de ARNcd para o gene b (Figura 1A). Várias linhas de tester eyFLP-marcm para recombinação no X, 2L, 2R, 3L e 3R foram estabelecidas e estão disponíveis dentro da comunidade mosca. 1. O manuseamento Fly, cruzes, e estadiamento <ol><li> Expandir o estoque marcm testador adequado à temperatura ambiente, preenchendo múltipla bottles ao mesmo tempo. Virar os adultos em garrafas frescos a cada 2-3 dias para que as virgens suficientes podem ser recolhidos para cruzamentos subsequentes. NOTA: Quando recolher virgens, evitar uma exposição prolongada ao CO 2, pois isso compromete a fecundidade das fêmeas. </li><li> Dependendo da escala do experimento, configurar cruzes mosca em frascos utilizando pelo menos 10 marcm virgens testador e 4 machos (por exemplo, para geração de imagens de repórteres fluorescentes e imunocoloração) ou em frascos com pelo menos 30 virgens e 10 homens (por exemplo, para isolamento de ARN e quantificação de invasividade). </li><li> Elevar progénie a 25 ° C sob um dia longo de 16 h / 8 h ciclo de luz-escuridão. Virar pais em novos frascos / garrafas a cada 24 horas para facilitar o preparo reprodutível de larvas e para evitar a superlotação. NOTA: Começando no dia 6 após a postura dos ovos (AEL), o terceiro controle instar alimentação das larvas parada final, começar vagando e entrar pupariation. Em contraste, o início da transição larva-pupa pode ser deDefiniu ou inexistente em larvas com EAD portadores de tumores clonais hiperplásicas ou malignos. </li></ol> 2. Coleta de terceiro instar larvas <ol><li> Para imunocoloração, recolher cerca de 20 final de larvas terceiro (vagando na parede e os de um tamanho corporal comparável a partir do alimento). Use uma pinça para escolher cuidadosamente as larvas do frasco ou garrafa e transferi-los para um prato de embrião de vidro cheio com solução salina tamponada com fosfato (PBS). <ol><li> Para o isolamento de ARN e a quantificação da capacidade de invasão, recolher, pelo menos, 80 final de larvas terceiro (vaguear sobre a parede e as de um tamanho comparável a partir do corpo de alimentos). Use uma garrafa de esguicho para esguichar PBS em uma garrafa mosca contendo larvas até que a superfície é coberta. </li><li> Suavizar as camadas superiores de alimento com uma espátula e despeje o purê alimentos que contenham larvas em uma placa de Petri. Utilizando uma pinça, gentilmente pegar larvas e transferi-los para um prato de embrião de vidro cheio de PBS. </li></ol></li><li> Lavar com PBS larvas until todos os alimentos residual é removido. Inspecionar as larvas sob o estereomicroscópio fluorescente com 8 a ampliação de 16x para descartar todos &quot;leopardo larvas&quot; que carregam manchas GFP positivas aleatórias em todo o corpo larval (Figura 2A, 2B e ver seção de discussão). NOTA: Para o isolamento de ARN incluem um passo de lavagem no penúltimo EtOH a 70% durante 1 min. </li><li> Coloque o prato contendo larvas selecionado na PBS em gelo até dissecção. larvas processo no prazo de 30 min para evitar efeitos adversos de frio e fome. </li></ol> 3. Dissecção da Larvas <ol><li> Para dissecar o EAD sob um microscópio estereoscópico, use um par de pinças para pressionar levemente a parte do meio da larva contra o fundo prato de vidro. Com a segunda pinça, pegue os ganchos boca larval e puxá-los para fora do corpo (Figura 2C). NOTA: A força de tracção é muitas vezes suficiente para quebrar os caules óptica que ligam o EAD pair para os lobos cerebrais. Se o cérebro ou suas partes ficar ligado a EAD e a sua presença não é desejada para outras aplicações, cortar os caules óptica com a ajuda dos dois pares de pinças. Para tumores invasivos (por exemplo, ras V12 Scrib 1) a conexão entre EAD e os lóbulos cerebrais torna-se cada vez mais obscurecido com o tempo por overgrowing e migração de células clonais. <ol><li> Para preparar o complexo de EAD / cérebro com um cordão nervoso intacto ventral (VNC) (Figura 2D), utilizar uma pinça para cortar uma larva no meio do corpo. Descarte a metade posterior. </li><li> Segurar a metade anterior do corpo de larvas entre as pontas de um fórceps, inverter a larva de dentro para fora, empurrando os ganchos boca com a ponta do fórceps e as segundas por laminagem a cutícula por cima com o primeiro par de fórceps. </li></ol></li><li> Utilizando uma pinça remova cuidadosamente todos os tecidos estranhos (por exemplo, glândulas salivares, gordura corporal e intestinal), deixando o par EAD ouo complexo EAD / cérebro ligado aos ganchos boca. Separar os ganchos na boca da cutícula sobrejacente. Livre o VNC, cortando as projeções axonal estendendo-se até os músculos do corpo e epiderme (Figuras 1B, 2C, 2D). NOTA: Os ganchos boca para permitir a manipulação segura do tecido com uma pinça e facilitar o afundamento mais eficiente e fácil reconhecimento de tecido durante a lavagem. Dissecados morfologia mudança EAD relativamente rápido quando mantidos unfixed em PBS. Limitar o tempo de dissecção para 20-30 min. </li><li> Para transferir o tecido, revestimento a ponta da micropipeta P20 pipetando o corpo restante carcaças várias vezes para cima e para baixo. Para transferir os complexos maiores EAD / cerebrais, corte a ponta da micropipeta P20 com uma tesoura. NOTA: O procedimento de revestimento é crítico para evitar a aderência e a perda de tecido dissecados durante a transferência. Uso de uma micropipeta P20 minimiza a transferência de fosfato em solução fixadora, limitando a diluição indesejada. <ol><li> Para imunocoloração, transferirEAD dissecado em um tubo de 0,5 ml cheio com 400 ul de paraformaldeido a 4% (PFA) fixador. Use 1,5 ml tubo com 1 ml PFA para ampliar EAD complexos cerebrais / significou para marcação de invasão. CUIDADO: PFA é altamente tóxico. Usar luvas e vestuário de protecção. Evitar o contacto com a pele, olhos ou membranas mucosas. </li><li> Para o isolamento do RNA transferir o EAD dissecado em um novo prato de vidro cheio de PBS frio. Use uma pinça para limpar EAD a partir das glândulas linfáticas e do anel, para minimizar a contaminação da amostra. </li><li> Retire a EAD dos ganchos na boca, cortando a ligação entre os discos de antena e boca ganchos usando uma pinça (Figura 2E). Para evitar RNA degradação e artefatos de expressão gênica, limitar o tempo de dissecção e 30 min. Siga para a etapa 7. Nota: Um investigador experiente pode dissecar cerca de 60 larvas em 30 min Tempo de exclusão necessários para a coleta de larvas e de lavar roupa. Total de rendimentos de ARN depender do tamanho EAD que variam entre os genótipos e DevelopmentAl etapas. Dissecação de 80-100 pares de controle de EAD de larvas terceira final deve render 5-8 ug de RNA total. </li></ol></li></ol> 4. Fixação, imunocoloração e montagem <ol><li> Fix amostras em PFA fixador durante 25 min, enquanto se nutação à temperatura ambiente. O tempo de fixação pode ser prolongado até 60 minutos sem alterar a morfologia do tecido e a antigenicidade de proteínas. </li><li> Retirar as amostras de agente de fixao e lavagem com PBST (PBS com 0,1% de Triton X-100), três vezes durante 10 min, utilizando um nutator. Utilizar um volume suficiente de PBST durante cada passo de lavagem (400 uL / ​​tubo de 0,5 ml). NOTA: Fixo complexos / cérebro EAD significou para marcação de invasão não necessitam de imunocoloração. sinal de GFP continua bem visível após a fixação. Avance para o passo 4.4 para a dissecção final. <ol><li> Para a imunocoloração, tecido bloco em 200 ul de uma solução de bloqueio (PBST com 0,3% de BSA) durante 20 min com agitação suave à temperatura ambiente. <ol><li> Incube com primary anticorpos diluídos em 100 ul de uma solução de bloqueamento durante a noite a 4 ° C enquanto agitando suavemente. Referem-se a folha de dados anticorpo primário ou a literatura publicada para a diluição de anticorpo recomendada. NOTA: A diluição de anticorpo primário pode precisar ser otimizado. </li><li> Após cinco lavagens de 10 min em PBST, tecido mancha com os anticorpos secundários fluorescentes diluído em solução de bloqueio, enquanto agitando durante 2 horas à temperatura ambiente ou durante a noite a 4 ° C no escuro. </li><li> Lavar as amostras três vezes 10 minutos em PBST. </li></ol></li></ol></li><li> Substituir PBST com 500 ul de solução DAPI-coloração. Para marcar F-actina, adicionar faloidina conjugada com um corante fluorescente para solução DAPI-coloração. Depois de 15 min no escuro nutação, lavar o tecido uma vez durante 10 min com PBST. NOTA: rotulagem F-actina pode ser realizada de forma independente da coloração com DAPI. </li><li> Para a etapa de dissecção final, transferir o tecido de volta em um prato de vidro PBS-cheia usando um pi 1 mlpette. Utilizando os dois pares de pinças de clipe para fora dos ganchos boca. <ol><li> Separa-se os cérebros que serão usados ​​para a quantificação da invasividade do EAD cortando a haste óptica como EAD coberto pode dificultar a análise. </li></ol></li><li> Colocar uma gota de meio de montagem (15 ul de uma lamela x 22 mm 22 mm) sobre uma corrediça objectivo. Com a ajuda de fórceps dicas, distribuir o meio numa camada fina. Transferir EAD, cérebros ou os complexos EAD / cerebrais para o meio de montagem com uma micropipeta P20. </li><li> Use as dicas fórceps ou uma haste de tungstênio para distribuir tecidos e endireitar o VNC no slide. </li><li> Para evitar a formação de bolhas durante a montagem, o primeiro toque de uma borda de uma lamela para o meio de montagem e, em seguida, lentamente inferior para o meio de montagem com a ajuda de fórceps. Use pequenos pedaços de papel de filtro ou lenço de papel para absorver o excesso de meio de montagem em torno das bordas lamela. NOTA: DABCO / poli (álcool vinílico) de montagem 4-88 endurece médiasa 4 ° C no decurso de uma hora. As lâminas podem ser armazenadas durante meses a 4 ° C. </li></ol> 5. Imagem Confocal <ol><li> Adquirir seções confocal individuais e pilhas usando um microscópio confocal equipado com 20X, objetivos petróleo 40X e 60X. </li><li> Evitar a saturação pixel. Use mesmos parâmetros de aquisição de imagem, incluindo a resolução da imagem, potência de entrada laser, ganho, offset, quadro de média, um tamanho de passo em uma série z e manter essas configurações para todos os genótipos para permitir comparações de intensidades de pixel entre os diferentes genótipos 21,22. NOTA: Para a visualização do complexo / cérebro EAD, gerar projeções máximas e costurar respectivas imagens, vizinhos. Para a preparação da imagem final, use pós software de processamento de imagem para a montagem do painel e para ajustar o brilho e contraste. </li></ol> 6. A quantificação de invasividade tumoral <ol><li> Definir um sistema de pontuação que vai caracterizar os diferentes níveis de invasivenes tumoraiss considerando o padrão espacial de invasão e a quantidade de células positivas para GFP espalhar para o cérebro e VNC. Prepare fichas de avaliação para documentação (Figura 3A). </li><li> Mount pelo menos 80 fixa cérebros intactos para cada genótipo em um slide utilizando 40 ul do meio de montagem por 24 milímetros x 50 mm lamela. <ol><li> Para permitir pontuação imparcial, pergunte a um partido neutro anonimizar os slides, para que o procedimento de pontuação é imparcial. Avaliar lâminas anónimos por dois membros do laboratório de forma independente. Divulgar genótipos somente após a contagem estiver concluída. </li></ol></li><li> Avaliar o grau de malignidade por um placar cega de cérebros montados sob um microscópio estereoscópico fluorescente equipado com um conjunto de filtros GFP. Use um marcador para rotular cérebros que já foram vistos a fim de evitar a dupla contagem (Figura 3B). </li><li> Calcula-se a percentagem de cérebros de queda em cada uma das categorias definidas por genótipo invasivos. Determinar os s estatísticosignificance usando um teste qui-quadrado (Figura 3C). </li></ol> 7. Isolamento RNA, DNase Tratamento e Verificação da Qualidade NOTA: Todos os reagentes (soluções, utensílios de plástico) utilizado nos passos seguintes deve ser isento de actividade de RNase. Use sempre luvas e usar um capuz química para o trabalho com solventes orgânicos. <ol><li> Transfira a EAD limpo com uma ponta de micropipeta P20 revestido em 1,5 ml tubo. </li><li> Deixe dissipador de tecido para o fundo do tubo, cuidadosamente remover PBS e substituí-la com 100 ul de reagente de lise de ARN (suficiente para até 140 DAE). </li><li> Lyse tecido em vórtex. Neste ponto, as amostras podem ser congelados em azoto líquido e armazenado a -80 ° C ou directamente processada com um protocolo de isolamento de ARN padrão. NOTA: As amostras de genótipos similares de várias rodadas de dissecação pode ser agrupada vez em reagente de lise RNA. </li><li> Encher o volume da amostra de 0,9 ml, com reagente de lise de ARN. Adicionar 0,2 ml de clorofórmio e misturar amostras Vigorously durante 15-20 seg. </li><li> Após 2 a 3 minutos de incubação à temperatura ambiente, centrifugar as amostras a 10.000 xg durante 15 min a 4 ° C. </li><li> ARN de transferência superior incolor fase aquosa contendo em um novo tubo, sem perturbar a interfase. Fique longe da interfase, pois contém proteínas e DNA genômico. NOTA: O volume recuperado deve ser de cerca de 60% do volume de reagente de lise de ARN usado para a homogeneização. </li><li> Precipitar o ARN por adição de 0,5 ml de álcool isopropílico. Vortex e incubar as amostras à temperatura ambiente durante 10 min. </li><li> Centrifugar as amostras a 10.000 xg durante 15 min a 4 ° C. </li><li> Descartar o sobrenadante e lavar o sedimento de ARN, uma vez com 600 ul de etanol a 75%. Depois de um curto vórtice e centrifugação (10.000 xg durante 7 minutos a 4 ° C) descartar todo o etanol e deixar o sedimento de ARN de ar seco durante 5-10 minutos colocando o tubo aberto em um tecido limpo limpar. NOTA: O sedimento de ARN pode ser visível como uma pequena branco opaco r /maduro na parte inferior do tubo ou invisível. As gotas de etanol restantes podem ser removidos cuidadosamente com ponta P10 micropipeta. </li><li> Dissolve-se o ARN em 50 jil de água tratada com DEPC em vortex ou solução que passa algumas vezes através de uma ponta de pipeta. </li><li> Para minimizar a contaminação com ADN genómico, tratar de ARN total com ADNase pela adição de 50 ul de uma mistura contendo 1 ul de ADNase (2 U / ul) e 10 ul de tampão de ADNase 10x em água tratada com DEPC. Vortex e girar para baixo. </li><li> Incubar as amostras a 37 ° C num banho de água ou de um bloco de aquecimento durante 30 a 40 min. Após a incubação, adicionar 100 ul de água tratada com DEPC para cada amostra. </li><li> Para inactivar a actividade enzimática e RNA limpo de ADNase, adicionar 200 ul de fenol: clorofórmio: álcool isoamílico (25: 24: 1) soluções, vortex durante 1 min e centrifuga-se as amostras a 10.000 xg durante 7 minutos a 4 ° C. </li><li> Transferir cuidadosamente a fase aquosa superior contendo ARN para um tubo fresco. Adicionar um volume igual (200 ul)de clorofórmio, misturar e repetir o passo de centrifugação a partir de cima. </li><li> Recolher cuidadosamente a fase superior e precipitar o ARN por adição de 1/10 do volume de 3 M de acetato de sódio, pH 5,2, preparada em DEPC H2O e 2,5 volumes de etanol a 100%. Misture bem em vórtice. NOTA: A adição de 0,5 ul de glicogénio (20 mg / mL) a uma mistura de precipitação ajuda a visualizar o sedimento de ARN. Para minimizar a perda de ARN, usar siliconada, tubos de ligação de baixa microcentrífuga de 1,5 ml. </li><li> Precipitar o RNA a -20 ° C durante pelo menos 1 h. NOTA: A incubação pode ser realizada durante a noite. As amostras podem ser colocadas a -80 ° C, bem. </li><li> Sedimento de ARN por centrifugação a 10000 xg durante 30 min a 4 ° C. Lave e pellet seco seguindo a descrição em 7,9. </li><li> Dissolve-se o ARN em 10-15 ul de água tratada com DEPC. ARN armazenar a -80 ° C. </li><li> Determinar a quantidade e a pureza do RNA através da medição OD a 230 nm, 260 nm e 280 nm utilizando um espectrofotómetro. Calcule RNA concentration, aplicando a convenção de que 1 DO a 260 nm é igual a 40 ug / ml de ARN. NOTA: O rácio A 260/280 de ARN &quot;pura&quot; é igual a 2,0, enquanto uma razão A 260/230 deve estar na gama de 2,0-2,2. As amostras de ARN com um A 260/280 razão entre 1,7 e 2,0 são adequados para aplicações a jusante, tais como a síntese de cDNA. Para a preparação de bibliotecas de ARNm-seq a qualidade e quantidade de ARN deve ser verificada utilizando um sistema de electroforese automatizada. A proporção 28S / 18S deve estar acima de 1,8. </li></ol>

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The authors declare that they have no conflict of interest.

As técnicas para gerar mosaicos genéticos em Drosophila estão entre as ferramentas mais sofisticadas para analisar e manipular a função do gene 33. O sistema eyFLP-marcm provou poderoso e robusto, uma vez que permite a indução do visualmente marcados, clones geneticamente definidas de uma maneira espacialmente restrita, isto é, em tecidos em que o potenciador é activa sem olhos 9,18. Isto é particularmente importante quando múltiplas lesões genéticas são com...

Cancer representa um dos grupo mais geneticamente heterogêneo de doenças, cuja incidência e mortalidade está aumentando dramaticamente, especialmente entre os idosos em todo o mundo. Cancer origem clonal de uma célula de iniciação tumor que escapa mecanismos supressores tumorais inerente e divide fora de controle. A acumulação gradual de lesões genéticas que cooperativamente promover o crescimento, a proliferação e a motilidade, enquanto inibir a morte e a diferenciação transforma o crescimento excessivo benigno inicial em um tumor altamente maligno metastático e mortal. Tornou-se evidente que, além de alterações genéticas, a progressão do tumor requer alterações no estroma circundante e diafonia entre os tipos de células tumorais e múltiplos (por exemplo, fibroblastos, células endoteliais, imunes e) no seu microambiente. Compreender os princípios moleculares subjacentes transformação maligna incluindo interações tumor-estroma é de grande importância para o desenvolvimento de prevenestratégias de e rastreio precoce, bem como tratamentos novos e eficazes para lutar contra a metástase do câncer e resistência aos medicamentos. A mosca da fruta Drosophila melanogaster tornou-se um sistema atraente para a investigação do cancro 1-4 devido ao seu tempo de geração rápido, notável conservação da sinalização nós entre moscas e seres humanos, redundância genética limitada e riqueza de ferramentas genéticas avançadas que facilitam a manipulação de quase qualquer gene em de maneira temporária e espacialmente restritos. Tumores geneticamente definidas de diferentes malignidade pode ser reprodutivelmente engenharia em Drosophila através da introdução de gain- e mutações de perda de função em um subconjunto de células progenitoras num tecido de outra forma tipo selvagem utilizando a técnica marcm 5. A ferramenta combina marcm FLP / FRT (FLP recombinase / FLP Reconhecimento Target) mediada por recombinação mitótica 6 com FLP-out 7 e Gal4 / UAS (Upstream Activation Sequence) gene alvo 8sistemas de expressão de 9. Com esta expressão método de qualquer transgene UAS-base, incluindo oncogene ou ADNc de proteína fluorescente ou repetições de ADN invertidos para silenciamento de genes induzido por ARNcd, será restrita a um clone de células que perderam um locus genético específico e um repressor Gal4 devido à recombinação (Figura 1A). Remendos clonais marcados com fluorescente verde (GFP) ou proteínas fluorescentes vermelho (por exemplo, RFP, DsRed, mCherry) pode ser facilmente rastreada ao longo do desenvolvimento, isolados e analisados. Importantemente, o seu comportamento pode ser directamente comparada com o tecido adjacente de tipo selvagem. Assim, questões pertinentes à célula efeitos autónomos e não autónomos de lesões genéticas podem ser convenientemente estudado. Semelhante a mamíferos, apenas clones em que múltiplas lesões oncogênicos são combinados se tornem malignas em Drosophila e recapitular características principais de câncer de mamíferos. Eles overproliferate, fugir apoptose, induzir a inflamação, tornar-se imortal e invasiva, em última análise, matando o hospedeiro 10-17. Aqui, descrevemos um protocolo para gerar tumores clonais geneticamente definidas no tecido ocular / antenas e do cérebro de larvas de Drosophila usando a técnica marcm. O método baseia-se um testador de estoque marcm que expressa a recombinase FLP de levedura sob o controlo do intensificador sem olhos (eyFLP) 18,19. Desta forma, os clones marcado com GFP são geradas em ambos epitélio colunar e peripodial do EAD e o neuroepitélio do cérebro ao longo estágios embrionários e larval (Figura 1a, b e de referência 20). Clones podem ser facilmente seguido até a idade adulta como a EAD desenvolve no olho adulto, antena e cabeça cápsula enquanto o neuroepitélio dá origem a neuroblastos que produzem neurônios do lóbulo óptica diferenciadas. Para facilitar a extensa caracterização molecular, funcional e fenotípica do tecido mosaico, que deescriba um protocolo para dissecção da EAD e do cérebro a partir do terceiro larvas e delinear a forma de processá-los por três aplicações diferentes: (i) a detecção de repórteres fluorescentes transgênicas e imunocoloração, (ii) quantificação de invasão tumoral e (iii) a análise dos altera a expressão do gene utilizando uma quantitativa PCR em tempo real (qRT-PCR), ou um ARNm de alto rendimento de sequenciação (ARNm-SEQ) (Figura 1C). O protocolo de imunocoloração pode ser usado para visualizar qualquer proteína de interesse com um anticorpo específico. Transgenic repórteres fluorescentes transcricionais fornecer informações espaço-temporal conveniente e precisa sobre a actividade de uma via de sinalização particular. repórteres celular específicos de linhagem, por outro lado, reflectem alterações qualitativas e quantitativas em populações de células no interior do tecido do mosaico e entre os tumores de diferentes genótipos. A quantificação do comportamento invasivo facilita a comparação de malignidade do tumor entre o genótipos. Finalmente, o protocolo descrevendo recolha e tratamento de EAD mosaico para o isolamento de RNA é adequado tanto para aplicações a jusante-pequena e grande escala, tais como a transcrição reversa seguida de qRT-PCR e mRNA-seq genome-wide, respectivamente. Os dados qualitativos e quantitativos obtidos a partir destes ensaios de fornecer novos insights sobre o comportamento social dos tumores clonais. Além disso, eles produzem uma base sólida para estudos funcionais sobre o papel dos genes individuais, redes genéticas e microambiente do tumor em diferentes fases e aspectos da tumorigénese.

<table><tbody><tr><td>Agar</td><td>Gew&amp;uuml;rzm&amp;uuml;hle Brecht, Eggenstein, Germany</td><td>00262-0500</td><td>Fly food recipe: Prepare 20 L fly food with 160 g agar, 360 g yeast, 200 g soy flour, 1.6 kg yellow cornmeal, 1.2 L malt extract, 300 ml light corn syrup, 130 ml propionic acid and 200 ml 15% nipagin. Fly food should be cooked for 1 hour at 85&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Corn syrup</td><td>Grafschafter Krautfabrik Josef Schmitz KG, Meckenheim, Germany</td><td>01939</td><td></td></tr><tr><td>Propionic acid</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>6026.1</td><td></td></tr><tr><td>Cornmeal</td><td>ReformKontor GmbH, Zarrentin, Germany</td><td>4010155063948</td><td></td></tr><tr><td>Malt extract</td><td>CSM Deutschland GmbH, Bremen, Germany</td><td>728985</td><td></td></tr><tr><td>Soy flour</td><td>Stockmeier Food GmbH, Herford, Germany</td><td>1000246441010</td><td></td></tr><tr><td>Yeast</td><td>Werner Ramspeck GmbH, Schwabach, Germany</td><td>210099K</td><td></td></tr><tr><td>Methyl-4-benzoate/ Nipagin</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>H5501</td><td>Prepare a 15% stock solution with 70% EtOH</td></tr><tr><td>Drosophila fly food vials</td><td>Kisker Biotech, Steinfurt, Germany</td><td>789008</td><td></td></tr><tr><td>Vial plugs</td><td>K-TK e.K., Retzstadt, Germany</td><td>1002</td><td></td></tr><tr><td>Drosophila fly food bottles</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>960177</td><td></td></tr><tr><td>Bottle plugs</td><td>K-TK e.K., Retzstadt, Germany</td><td>1002 S</td><td></td></tr><tr><td>Poly(vinyl alcohol) 4-88/[-CH&lt;sub&gt;2&lt;/sub&gt;CHOH-]&lt;sub&gt;n&lt;/sub&gt;</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>81381</td><td>Mounting medium recipe: Dissolve 6 g Poly(vinyl alcohol) 4-88 in 39&amp;nbsp;ml Millipore H&lt;sub&gt;2&lt;/sub&gt;O, 6 ml 1 M Tris (pH 8.5) and 12.5 ml glycerol. Stir overnight at 50&amp;nbsp;&amp;deg;C and centrifuge 20&amp;nbsp;min at 5,000 rpm. Add DABCO to the supernatant to get a final concentration of 2.5%. Store aliquots at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>1,4-Diazabicyclo[2.2.2]octane/ DABCO&amp;nbsp;</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>D2522</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>9002-93-1</td><td></td></tr><tr><td>Phosphate-buffered saline/ PBS</td><td></td><td></td><td>137 mM NaCl, 2.7 mM KCl, 10 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 1.8 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, pH 7.4 in Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>PBST</td><td></td><td></td><td>0.1% Triton X-100 in PBS</td></tr><tr><td>Paraformaldehyde/ PFA</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>158127</td><td>4% PFA fixative recipe: Dissolve 4&amp;nbsp;g PFA in 80 ml Millipore H&lt;sub&gt;2&lt;/sub&gt;O on a magnetic stirrer plate heated to 55&amp;nbsp;&amp;deg;C. Add 1 M NaOH dropwise until all PFA particles are dissolved. Add 10 ml 10x PBS and adjust the pH to 7.4 with 1 M HCl. Mix in 100 &amp;micro;l Triton X-100 and fill up with Millipore H&lt;sub&gt;2&lt;/sub&gt;O to 100 ml. Store aliquots at -20 &amp;deg;C. Avoid repeated thawing. (CAUTION: PFA is highly toxic. Prepare the PFA fixative in a fume hood. Wear a self-contained breathing apparatus, gloves and clothing. Avoid contact with skin, eyes or mucous membranes.)</td></tr><tr><td>Bovine Serum Albumin/ BSA</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>A3059</td><td>Blocking solution recipe: Dissolve 0.3% BSA in PBST</td></tr><tr><td>Fluorescently labeled phalloidin</td><td>Invitrogen, Karlsruhe, Germany, Molecular Probes</td><td>A12379, A22283, A22284</td><td>Phalloidin stock solution: Dissolve powder (300 U) in 1.5 ml Methanol. Store at -20 &amp;deg;C. For staining, use in dilution 1:250 (A12379, A22283) and 1:100 (A22284) in PBST.</td></tr><tr><td>4&amp;rsquo;,6-Diamidino-2-phenylindol Dihydrochlorid/ DAPI</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>6335</td><td>DAPI staining solution: Prepare a stock solution of 5 mg/ml in Millipore H&lt;sub&gt;2&lt;/sub&gt;O and store aliquots at 4 &amp;deg;C. Used in dilution 1:1,000 in PBST.</td></tr><tr><td>Mouse anti-H2 antibody</td><td>Kurucz et al., 2003</td><td></td><td>Used in dilution 1:500 in blocking solution</td></tr><tr><td>Cy5 AffiniPure Donkey Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch, Suffolk, UK</td><td>715-175-151</td><td>Used in dilution 1:500 in blocking solution</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools, Heidelberg, Germany</td><td>11295-10</td><td></td></tr><tr><td>Glass embryo dish (30&amp;nbsp;mm)</td><td>Thermo Scientific, Schwerte, Germany</td><td>E90</td><td></td></tr><tr><td>Tungsten needles</td><td>Fine Science Tools, Heidelberg, Germany</td><td>10130-20</td><td></td></tr><tr><td>Nickel Plated Pin Holder</td><td>Fine Science Tools, Heidelberg, Germany</td><td>26018-17</td><td></td></tr><tr><td>Microscope slides</td><td>VWR, Darmstadt, Germany</td><td>631-1553</td><td></td></tr><tr><td>Coverslips 22 x 22 mm (#1 Menzel-Gl&amp;auml;ser)</td><td>VWR, Darmstadt, Germany</td><td>631-1336</td><td></td></tr><tr><td>Coverslips 24 x 50 mm (0.13-0.16&amp;nbsp;mm)</td><td>Thermo Scientific, Schwerte, Germany</td><td>1076371</td><td></td></tr><tr><td>Kimtech Science Precision Wipes</td><td>Thermo Scientific, Schwerte, Germany</td><td>06-677-70</td><td></td></tr><tr><td>Dissecting stereomicroscope</td><td>Olympus, Hamburg, Germany</td><td>SZX7 (DF PLAPO 1X-4 Japan)</td><td></td></tr><tr><td>Fluorescent stereomicroscope</td><td>Olympus, Hamburg, Germany</td><td>SZX16 (SDF PLAPO 0.8X Japan) with DP72 CCD camera</td><td>Equipped with the narrow blue bandpass filter set for excitation of GFP other blue excitable fluorochromes (excitation filter BP460-480 nm, barrier filter BA495-540 nm) and narrow green excitation and longpass barrier filter for RFP and other green excitable fluorochromes (excitation filter BP530-550, barrier filter BA575IF). Software: Olympus cellSens Standard 1.11.</td></tr><tr><td>Confocal microscope</td><td>Olympus, Hamburg, Germany</td><td>FV1000</td><td>Equipped with inverted IX81 microscope. Objectives: 20&amp;times; UPlan S-Apo (NA 0.85), 40&amp;times; UPlanFL (NA 1.30) and 60&amp;times; UPlanApo (NA 1.35). Lasers: UV laser Diode LD405 (50&amp;nbsp;mW), Argon laser, multi-line 457/(476)/ 488/515 (40&amp;nbsp;mW), Yellow/Green laser diode LD560 (15&amp;nbsp;mW) and Red Laser diode 635 (20&amp;nbsp;mW). Software: Fluoview 2.1c Software</td></tr><tr><td>Drosophila cooled incubator</td><td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td><td>&amp;nbsp;Sanyo MIR553</td><td></td></tr><tr><td>Squirt bottle</td><td>VWR, Darmstadt, Germany</td><td>215-8105</td><td></td></tr><tr><td>BD Clay Adams Nutator Mixer</td><td>VWR, Darmstadt, Germany</td><td>15172-203</td><td></td></tr><tr><td>ELMI Digital Rocking Shaker</td><td>VWR, Darmstadt, Germany</td><td>DRS-12</td><td></td></tr><tr><td>5 PRIME Isol-RNA Lysis Reagent</td><td>VWR, Darmstadt, Germany</td><td>2302700</td><td>The protocol is compatible with other TRIzol-based reagents, &lt;em&gt;e.g.&lt;/em&gt;, TRIreagent from Sigma (Cat. Nr.T9424), TRIzol reagent from Thermofisher (Cat. Nr. 15596).</td></tr><tr><td>Diethyl pyrocarbonate/ DEPC</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>D5758</td><td>Dilute DEPC 1:1,000 in Millipore H&lt;sub&gt;2&lt;/sub&gt;O, stir overnight and autoclave.</td></tr><tr><td>UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1)</td><td>ThermoFischer Scientific, Invitrogen, Karlsruhe, Germany</td><td>15593-031</td><td></td></tr><tr><td>Chloroform</td><td>Merck, Darmstadt, Germany</td><td>102445</td><td></td></tr><tr><td>TURBO DNase (2 U/&amp;micro;l)</td><td>Thermo Scientific, Schwerte, Germany</td><td>AM2238</td><td></td></tr><tr><td>Invitrogen UltraPure Glycogen</td><td>ThermoFischer Scientific, Invitrogen, Karlsruhe, Germany</td><td>10-814-010</td><td></td></tr><tr><td>Sodium acetate trihydrate</td><td>VWR, Darmstadt, Germany</td><td>27652.232</td><td>Prepare a 3 M Sodium acetate solution with DEPC-H&lt;sub&gt;2&lt;/sub&gt;O and adjust the pH to 5.2</td></tr><tr><td>2-Propanol</td><td>Merck, Darmstadt, Germany</td><td>109634</td><td></td></tr><tr><td>Ethanol</td><td>Merck, Darmstadt, Germany</td><td>100983</td><td>Prepare a 75% EtOH dilution with DEPC-H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>UV/Vis-Spectrophotometre NanoDrop ND-8000</td><td>Thermo Scientific, Schwerte, Germany</td><td>ND-8000</td><td></td></tr><tr><td>Eppendorf Microcentrifuge (Refrigerated)</td><td>Thermo Scientific, Schwerte, Germany</td><td>5417R</td><td></td></tr><tr><td>Experion RNA StdSens Analysis Kit</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>7007103</td><td></td></tr><tr><td>Experion Automated Electrophoresis Station</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>7007010</td><td></td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>Thermo Scientific, Schwerte, Germany</td><td>18080044</td><td></td></tr><tr><td>Oligo d(T) Primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>Prepare 100 &amp;micro;M stock solutions in DEPC-H&lt;sub&gt;2&lt;/sub&gt;O and store at -20 &amp;deg;C</td></tr><tr><td>dNTP Mixture</td><td>Takara Bio Europe/Clonetech, Saint-Germain-en-Laye, France</td><td>4030</td><td>Store aliquots of 25 &amp;micro;l at -20&amp;deg;C</td></tr><tr><td>CFX96 Touch Real-Time PCR Detection System</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>1855195</td><td></td></tr><tr><td>iQ SYBR Green Supermix</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>170-8882</td><td></td></tr><tr><td>Hard-Shell PCR Plates 96-well, thin wall</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>HSP9601</td><td></td></tr><tr><td>Microseal &#039;B&#039; Film</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>MSB1001</td><td></td></tr><tr><td>&lt;em&gt;rp49&lt;/em&gt; Forward primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; TCC TAC CAG CTT CAA GAT GAC 3&#039;</td></tr><tr><td>&lt;em&gt;rp49&lt;/em&gt; Reverse primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; CAC GTT GTG CAC CAG GAA CT 3&#039;</td></tr><tr><td>&lt;em&gt;mmp1&lt;/em&gt; Forward primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; AGG GCG ACA AGT ACT ACA AGC TGA 3&#039;</td></tr><tr><td>&lt;em&gt;mmp1&lt;/em&gt; Reverse primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; ACG TCT TGC CGT TCT TGT AGG TGA 3&#039;</td></tr></tbody></table>

the drosophila imaginal disc tumor model: visualization and quantification of gene expression and tumor invasiveness using genetic mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

Para demonstrar o potencial da técnica eyFLP-marcm para gerar manchas marcadas com GFP de genótipos definidos em Drosophila EAD, foram induzidos três tipos de clones: (1) controle somente expressando GFP, (2) tumores malignos expressar uma forma oncogénica do pequeno Ras (Ras V12) da proteína G em um fundo de perda homozigótica de um scribble gene supressor de tumor (Scrib 1), e (3) sobrecrescimento mas não invasivo ras V12...

Watch this Scientific Journal Video about The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics at JoVE.com

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Este protocolo demonstra como gerar tumores clonais fluorescente marcada, geneticamente definidas no Drosophila olho / discos imaginais antenares (DAE). Ele descreve como dissecar o EAD e do cérebro a partir do terceiro larvas e como processá-los para visualizar e quantificar as mudanças de expressão gênica e invasão tumoral.

o<em&gt; Drosophila</em&gt; Imaginal Modelo Disc Tumor: visualização e quantificação da expressão gênica e invasão tumoral Usando Mosaicos Genéticos

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

the-drosophila-imaginal-disc-tumor-model-visualization-quantification

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

11.0K visualizações.  University of Cologne.  Este protocolo demonstra como gerar tumores clonais fluorescente marcada, geneticamente definidas no Drosophila olho / discos imaginais antenares (DAE). Ele descreve como dissecar o EAD e do cérebro a partir do terceiro larvas e como processá-los para visualizar e quantificar as mudanças de expressão gênica e invasão tumoral. 

Vídeo: The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

Mosaic Zebrafish Transgénese para Análise Funcional Genomic de candidatos Genes de cooperação em Tumor Patogênese

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

Biologia do Desenvolvimento

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes.

Indução e diagnóstico de tumores em<em&gt; Drosophila</em&gt; Epitelio do disco imaginal

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

Pesquisa do câncer

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

Mapeamento e Aplicação de Enhancer-trap Expressão Flippase em larval e Adultos Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Neurociência

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Análise morfológica de Drosophila Larval sensorial periférica Dendrites Neuron e axônios Usando Mosaicos Genéticos

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

Um protocolo para indução genética e Visualização de tumores benignos e invasoras em Cephalic Complexos de<em&gt; Drosophila melanogaster</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

Medicina

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 ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Microdissecção a laser Aplicada ao perfil de expressão gênica de subconjunto de células a partir do Drosophila Asa Disco

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

Dissecção e Imunocoloração do Imaginário discos de<em&gt; Drosophila melanogaster</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

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 ...

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