Os autores reconhecem o apoio do NIH/NIGMS (R01GM084135 de HJN) e Fundação de Mizutani para eficientemente (grant #110071 para HJN) e é grato ao Tom V. Lee para discussões e sugestões sobre os ensaios e Spyros Artavanis-Tsakonas, Hugo Bellen, Robert Fleming, Ken Irvine e a drosófila genômica recurso Center (DGRC) para plasmídeos e linhas celulares.

1. preparação do RNA encalhado dobro (dsRNA) para Knockdown de Souza
<ol>
	<li>Amplificação por PCR dos produtos
	<ol>
		<li>Use o DNA genômico do selvagem-tipo branco amarelo (y w) e pAc5.1-EGFP como modelo e os seguintes pares de cartilha para amplificar os fragmentos de DNA usados na síntese de dsRNA. Use o seguinte perfil térmico de PCR: desnaturação (95 ° C, 30 s), recozimento (58 ° C, 30 s) e extensão (72 ° C, 1 min). 
		Reforçada a proteína verde fluorescente (EGFP) dsRNA Cartilhas (5' - 3')- Primeira demão para diante-GAAATTAATACGACTCACTATAGGGGGTGAGCAAGGGCGAGGAG Reverter a primeira demão-GAAATTAATACGACTCACTATAGGGGGTCTTTGCTCAGGGCGG 
		Shams dsRNA Cartilhas (5' - 3')- Primeira demão para diante-TTAATACGACTCACTATAGGGGAGATGCTGTATGTGGACACGGAT Reverter a primeira demão-TTAATACGACTCACTATAGGGGAGATCCGTGGATAACCTTAACGA Nota: EGFP dsRNA é usado como controle negativo.</li>
	</ol>
	</li>
	<li>Gel-purifica os produtos de reação em cadeia (PCR) polimerase usando um kit de purificação de gel de comerciais de acordo com o protocolo do fabricante.</li>
	<li>Execute em vitro transcrição usando um produto comercial capaz de transcrever de um promotor T7 de acordo com o protocolo do fabricante.</li>
	<li>Purificar o dsRNA usando um kit de purificação de RNA de acordo com o protocolo do fabricante e armazenar a-80 ° C.</li>
	<li>Avalie a eficiência de Souza knockdown usando as seguintes etapas (1.5.1-1.5.5).
	<ol>
		<li>Contagem das células manualmente usando um hemocytometer e placa 5 x 105 S2 células em cada poço de um 6 bem placa em 1 mL/bem do meio do Schneider suplementado com 10% soro Fetal bovino (FBS) e 100 U/mL de penicilina-estreptomicina.</li>
		<li>Adicione 7,5 µ g de EGFP- ou logros dsRNA a cada poço e incubar a 25 ° C por 24 h.</li>
		<li>Colheita do controle (EGFP dsRNA-tratados) e logros dsRNA-tratados S2 células pipetando gentil seguido de granulação para baixo por centrifugação a 1.000 x g por 5 min a 25 ° C e o processo de isolamento do RNA usando um kit de extração de RNA de acordo com protocolo do fabricante.</li>
		<li>Quantificar o RNA usando um espectrofotômetro e processo 100 ng do RNA para o 1 passo qRT-PCR utilizando reagentes comerciais qPCR e primer/sondas e o seguinte perfil térmico de PCR: desnaturação (95 ° C, 15 s) e recozimento/extensão (60 ° C, 1 min). Nota: Consulte a Tabela de materiais , para obter informações sobre os conjuntos de primer/sonda e o instrumento utilizado para experimentos de Souza e controle qRT-PCR nestes estudos.</li>
		<li>Calcule os níveis de mRNA relativo Souza usando o 2–ΔΔCT método16.</li>
	</ol>
	</li>
</ol>
2. avaliação da ligação entre o receptor do entalhe e Trans-ligantes
<ol>
	<li>Prepare o sinal de recepção células (S2 expressando o receptor do entalhe).
	<ol>
		<li>Contar as células manualmente usando um hemocytometer e placa de 5 x 105 S2 e estáveis células S2-entalhe em cada poço de uma placa de 6 em 1 mL/bem do meio do Schneider suplementado com 10% FBS e 100 U/mL de penicilina-estreptomicina. Nota: Para as células S2-entalhe, que estão disponíveis de Drosophila genómica Resource Center (DGRC), metotrexato 200 nM na mídia. Para preparar uma solução stock de metotrexato de 0,5 mM, primeiro prepare uma solução de metotrexato 20 mM em 250 µ l de 1 M de NaOH. Em seguida, diluir esta solução a 0,5 mM na solução salina tampão de fosfato 1 M e em-20 ° C. Nas células de S2-entalhe, expressão da proteína Notch está sob o controle do promotor metalotioneína Drosophila do vetor de PGTO .</li>
		<li>Adicione 7,5 µ g do dsRNA a cada poço e incubar a 25 ° C por 24 h.</li>
		<li>Adicione 0,7 mM CuSO4 para induzir a expressão de entalhe e incubar a 25 ° C por 3 dias. Nota: CuSO4 é usado para induzir a expressão de proteínas controlado pelo promotor metalotioneína.</li>
	</ol>
	</li>
	<li>Prepare o envio de sinal de células (célula S2 expressando Delta ou Serrate ligantes).
	<ol>
		<li>Contar as células manualmente usando um hemocytometer e placa ~ 5 x 106 estável S2-Delta ou S2-SerrateTom células em cada poço de uma placa de 6 em 1 mL/poço do meio do Schneider suplementado com 10% FBS e 100 U/mL de penicilina-estreptomicina. Nota: Adicione 200 metotrexato nM para células S2-Delta e 100 µ g/mL hptII para células S2-SerrateTom na mídia. Expressão do Delta e serrilhadoTom— uma versão funcional, tomate-Tag do serrilhado12— está sob Drosophila metalotioneína promotor do vetor de PGTO .</li>
		<li>Adicione 0,7 mM CuSO4 para induzir a expressão de ligantes e incubar a 25 ° C por 3 h.</li>
	</ol>
	</li>
	<li>Execute a agregação entre as células-envio de sinal e o sinal de recepção.
	<ol>
		<li>Colher o S2 dsRNA-tratados (controle) e células de S2-entalhe por pipetagem suave e placa de 2.5 x 105 células/poço em uma placa de 24 após a contagem manual por hemocytometer.</li>
		<li>Adicionar 5 x 105 estável Delta-S2 S2-SerrateTom células ou em um volume total de 200 µ l de meio do Schneider (suplementado com 10% FBS e 100 U/mL de penicilina-estreptomicina). Nota: Todas as células de S2 manipulação (incluindo tratamento de chapeamento, indução e dsRNA) foi feito sob uma armário de biossegurança.</li>
		<li>Coloque a placa em um agitador orbital a 150 rpm (942,48 rad/min).</li>
		<li>Depois de 1 min, misturar o conteúdo de cada poço e levar 20 µ l para contagem do número de agregados.<ol>
			<li>Ao mesmo tempo tome uma imagem representativa sob microscópio composto invertido usando ampliação de 10x (objetivo PLL 10/0.25).</li>
			<li>Contar manualmente os agregados (&#62; 6 células) usando um hemocytometer.</li>
		</ol>
		</li>
		<li>Coloque a placa de um agitador.</li>
		<li>Repita a aquisição da imagem e a contar depois de 5 min e 15 min de agregação.</li>
	</ol>
	</li>
	<li>Realizar a quantificação de trans-ligação.
	<ol>
		<li>Calcule o número de agregados por mL entre células S2 e S2-Delta ou S2-SerrateTom células como um controle de fundo.</li>
		<li>Calcular o número de agregados por mL entre entalhe em S2 e S2-Delta ou S2-SerrateTom células (magnitude de trans-vinculação).</li>
	</ol>
	</li>
</ol>
3.Avaliação da inibição da ligação entre o entalhe e Trans-ligantes por Cis-ligantes
<ol>
	<li>Prepare células de sinal de recepção.
	<ol>
		<li>Placa de 5 x 105 S2 células em cada poço de uma placa de 6 em 1 mL/bem do meio do Schneider suplementado com 10% FBS e 100 U/mL de penicilina-estreptomicina.</li>
		<li>Adicione 7,5 µ g do dsRNA a cada poço e incubar a 25 ° C por 24 h.</li>
		<li>Co transfect as células dsRNA-tratados com pBluescript e PMT-entalhe11 (DGRC) sozinho ou com Entalhe em PGTO e pMT-Delta11 (DGRC) ou pMT-Serrate17 para um total Concentração de DNA de 2 µ g/bem usando um reagente de transfeccao comercial de acordo com o protocolo do fabricante. Nota: pBluescript é usado como um controle. As células transfectadas co serão chamadas S2-entalhe &#38; Deltatransitória ou S2-entalhe &#38; Serratetransitória de células daqui por diante.</li>
		<li>Incube as células S2 transfectadas transitoriamente a 25 ° C por 24 h.</li>
		<li>Adicione 0,7 mM CuSO4 para induzir a expressão de entalhe e ligantes e incubar a 25 ° C por 3 dias.</li>
	</ol>
	</li>
	<li>Prepare o envio de sinal células conforme descrito no ponto 2.2.</li>
	<li>Execute a agregação entre as células de sinal-envio e recebimento de sinal conforme descrito na seção 2.3.</li>
	<li>Quantificar a inibição de trans-ligação por cis-ligantes.
	<ol>
		<li>Calcule o número de agregados por mL entre células S2 e S2-Delta ou S2-Serrate células como um controle de fundo.</li>
		<li>Quantificar a magnitude da inibição pelo cis-ligantes como segue.<ol>
			<li>Defina A como número de agregados entre S2-entalhetransitória e células S2-Delta.</li>
			<li>Definir B como número de agregados entre células S2 transitoriamente co transfected com entalhe e Delta (S2-entalhe &#38; Deltatransitória) e células S2-Delta.</li>
			<li>Calcular a agregação relativa C = (B x 100) /A</li>
			<li>Calcular a magnitude de inibição por cis- ligante (Delta ou Serrate) como 100 – C. Nota: como exemplo, se a agregação relativa é 45% e, em seguida, cis-ligantes são considerados para inibir o trans-vinculação de 55%.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>de Celis, J. F., Bray, S., Garcia-Bellido, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signalling+regulates+veinlet+expression+and+establishes+boundaries+between+veins+and+interveins+in+the+Drosophila+wing.">Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing.</a> Development. 124 (10), 1919-1928 (1997).</li><li>Fortini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling:+the+core+pathway+and+its+posttranslational+regulation.">Notch signaling: the core pathway and its posttranslational regulation.</a> Dev Cell. 16 (5), 633-647 (2009).</li><li>del Alamo, D., Rouault, H., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+significance+of+cis-inhibition+in+Notch+signalling.">Mechanism and significance of cis-inhibition in Notch signalling.</a> Curr Biol. 21 (1), R40-R47 (2011).</li><li>Sprinzak, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis-interactions+between+Notch+and+Delta+generate+mutually+exclusive+signalling+states.">Cis-interactions between Notch and Delta generate mutually exclusive signalling states.</a> Nature. 465 (7294), 86-90 (2010).</li><li>Kopan, R., Ilagan, M. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+canonical+Notch+signaling+pathway:+unfolding+the+activation+mechanism.">The canonical Notch signaling pathway: unfolding the activation mechanism.</a> Cell. 137 (2), 216-233 (2009).</li><li>Huppert, S. S., Jacobsen, T. L., Muskavitch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+regulation+is+central+to+Delta-Notch+signalling+required+for+Drosophila+wing+vein+morphogenesis.">Feedback regulation is central to Delta-Notch signalling required for Drosophila wing vein morphogenesis.</a> Development. 124 (17), 3283-3291 (1997).</li><li>Lee, T. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+regulation+of+notch+signaling+by+xylose.">Negative regulation of notch signaling by xylose.</a> PLoS Genet. 9 (6), e1003547 (2013).</li><li>Lee, T. V., Pandey, A., Jafar-Nejad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylosylation+of+the+Notch+receptor+preserves+the+balance+between+its+activation+by+trans-Delta+and+inhibition+by+cis-ligands+in+Drosophila.">Xylosylation of the Notch receptor preserves the balance between its activation by trans-Delta and inhibition by cis-ligands in Drosophila.</a> PLoS Genet. 13 (4), e1006723 (2017).</li><li>Jacobsen, T. L., Brennan, K., Arias, A. M., Muskavitch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis-interactions+between+Delta+and+Notch+modulate+neurogenic+signalling+in+Drosophila.">Cis-interactions between Delta and Notch modulate neurogenic signalling in Drosophila.</a> Development. 125 (22), 4531-4540 (1998).</li><li>Schneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines+derived+from+late+embryonic+stages+of+Drosophila+melanogaster.">Cell lines derived from late embryonic stages of Drosophila melanogaster.</a> J Embryol Exp Morphol. 27 (2), 353-365 (1972).</li><li>Fehon, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interactions+between+the+protein+products+of+the+neurogenic+loci+Notch+and+Delta,+two+EGF-homologous+genes+in+Drosophila.">Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila.</a> Cell. 61 (3), 523-534 (1990).</li><li>Fleming, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extracellular+region+of+Serrate+is+essential+for+ligand-induced+cis-inhibition+of+Notch+signaling.">An extracellular region of Serrate is essential for ligand-induced cis-inhibition of Notch signaling.</a> Development. 140 (9), 2039-2049 (2013).</li><li>Saj, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+ex+vivo+and+in+vivo+RNAi+screen+for+notch+regulators+in+Drosophila+reveals+an+extensive+notch+interaction+network.">A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network.</a> Dev Cell. 18 (5), 862-876 (2010).</li><li>Fiuza, U. M., Klein, T., Martinez Arias, A., Hayward, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+ligand-mediated+inhibition+in+Notch+signaling+activity+in+Drosophila.">Mechanisms of ligand-mediated inhibition in Notch signaling activity in Drosophila.</a> Dev Dyn. 239 (3), 798-805 (2010).</li><li>Ahimou, F., Mok, L. P., Bardot, B., Wesley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adhesion+force+of+Notch+with+Delta+and+the+rate+of+Notch+signaling.">The adhesion force of Notch with Delta and the rate of Notch signaling.</a> J Cell Biol. 167 (6), 1217-1229 (2004).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Okajima, T., Xu, A., Irvine, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+notch-ligand+binding+by+protein+O-fucosyltransferase+1+and+fringe.">Modulation of notch-ligand binding by protein O-fucosyltransferase 1 and fringe.</a> J Biol Chem. 278 (43), 42340-42345 (2003).</li><li>Acar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rumi+is+a+CAP10+domain+glycosyltransferase+that+modifies+Notch+and+is+required+for+Notch+signaling.">Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling.</a> Cell. 132 (2), 247-258 (2008).</li><li>Bruckner, K., Perez, L., Clausen, H., Cohen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosyltransferase+activity+of+Fringe+modulates+Notch-Delta+interactions.">Glycosyltransferase activity of Fringe modulates Notch-Delta interactions.</a> Nature. 406 (6794), 411-415 (2000).</li><li>Yamamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+EGF+repeat-8+of+Notch+discriminates+between+Serrate/Jagged+and+Delta+family+ligands.">A mutation in EGF repeat-8 of Notch discriminates between Serrate/Jagged and Delta family ligands.</a> Science. 338 (6111), 1229-1232 (2012).</li></ol>

Os autores não têm nenhum conflito de interesses.

Canônico de sinalização Notch depende das interacções entre o receptor do entalhe e seus ligantes5. Embora a maioria dos estudos sobre a via de Notch principalmente considerar a vinculação do entalhe e ligantes em células vizinhas (trans), entalhe e ligantes mesmo células interagem e estes so-called cis-interações podem desempenhar um papel inibitório no entalhe sinalização de3,4. Nesse sentido, para decifra...

Canônico de sinalização Notch é um mecanismo de comunicação de curto alcance de célula para célula que exige contato físico dos vizinhos células para facilitar a interação entre receptores Notch e seus ligantes1. Interação do receptor do entalhe (presente na superfície das células do sinal de recepção) com ligantes (presentes na superfície de sinal-enviando células) inicia sinalização Notch e é conhecida como trans-ativação2. Por outro lado, a interação entre entalhe e seus ligantes na mesma cela leva à inibição da via de entalhe de e é conhecida como cis-inibição3. O equilíbrio entre trans- e cis-interações é necessária para garantir o ideal Notch ligante-dependentes, sinalização4. Drosófila tem um receptor do entalhe e dois ligantes (Delta e Serrate) ao contrário de mamíferos, que têm quatro receptores Notch e cinco ligantes [irregulares 1 (JAG1), JAG2, delta-like 1 (DLL1), DLL3 e DLL4]5. Tendo esta simplicidade, o modelo de Drosophila oferece a facilidade para dissecar/estudo os efeitos dos modificadores de percurso em interações de entalhe-ligante e, posteriormente, na sinalização Notch. Em determinados contextos durante o desenvolvimento animal (incluindo desenvolvimento de asa em drosófila), ambos de cis- e trans-interações envolvidas para alcançar a sinalização adequada do entalhe e célula de destino1,6 . É importante distinguir os efeitos dos modificadores de via Notch nestes contextos na cis- e trans-interações de entalhe com seus ligantes.
Nosso grupo relatado anteriormente que a adição de um resíduo de hidrato de carbono chamado xilose de Drosophila entalhe negativamente regula entalhe sinalização em determinados contextos, incluindo asa desenvolvimento7. Perda de Souza (a enzima que xylosylates entalhe) leva a um fenótipo de "perda da veia da asa"7. Mais recentemente, experimentos de dosagem de gene e clonal análise foram utilizados para mostrar que perda de Souza realça recordando, mediada por Delta entalhe. Para distinguir se a sinalização reforçada de entalhe em mutantes de Souza é um resultado da diminuição da cis-inibição ou aumento trans-ativação, estudos de gravidez ectópica superexpressão de ligantes Notch em discos imaginária asa larval usando o driver de dpp-GAL4 foram realizadas. Estas experiências fornecidas provas sugerindo que Shams regula trans-ativação de entalhe por Delta sem afetar o entalhe cis-inibição por ligantes8. No entanto, feed-back regulamentos e efeitos de ligantes endógenos podem complicar a interpretação dos estudos de superexpressão ectópica1,6,9.
Para resolver esse problema, células de Drosophila S210 foram usados, que preveem um sistema simples em vitro entalhe-ligante interação estudos11,12. Células de S2 não express endógena do receptor do entalhe e Delta ligante11 e expressar um nível baixo de serrilhado,13, que não afeta o entalhe-ligante agregação experimentos8. Portanto, as células S2 podem estàvel ou transitoriamente transfected por entalhe e/ou ligantes individuais (Delta ou Serrate) para gerar células que expressam exclusivamente o receptor do entalhe ou um dos seus ligantes ou uma combinação deles. Mistura de células do entalhe-expressando S2 com ligante-expressando resultados de células S2 na formação de agregados heterotypic mediada pelo receptor-ligante ligação11,12,14. Quantificação da formação agregada fornece uma medida da trans-vinculação entre entalhe e seus ligantes15 (Figura 1). Da mesma forma, as células de S2 podem ser co transfectadas com ligantes Notch e Delta ou Serrate (ou seja, cis-ligantes). Cis-ligantes nestas células S2 entalhe-expressando revogar a vinculação do entalhe com trans-ligantes e resultado em diminuição da formação agregado8,12,14. A diminuição relativa na formação de agregados causada por cis-ligantes fornece uma medida do efeito inibitório do cis-ligantes na ligação entre o entalhe e trans-ligantes (Figura 2). Nesse sentido, os ensaios de agregação celular foram utilizados para examinar o efeito de perda de xylosylation trans- e cis-interações entre entalhe e seus ligantes.
Aqui, apresentamos um protocolo detalhado para ensaios de agregação de célula objetivou avaliar a vinculação do entalhe com trans-ligantes e sua inibição pelo cis-ligantes utilizando células de Drosophila S2. Como exemplo, podemos fornecer os dados que nos permitiram determinar o efeito de entalhe xylosylation na ligação entre o entalhe e trans-Delta8. Estes ensaios simples fornecem uma avaliação semi-quantitativa do entalhe-ligante interações em vitro e ajudam a determinar os mecanismos moleculares subjacentes os efeitos na vivo de modificadores de via Notch.

<table border="0" cellpadding="0" cellspacing="0" width="1127">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:415px;">BioWhittaker Schneider&#8217;s Drosophila medium, Modified</td>
			<td style="width:187px;">Lonza</td>
			<td style="width:131px;">04-351Q</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:415px;">HyClone Penicillin-Streptomycin 100X solution</td>
			<td style="width:187px;">GE Healthcare lifescience&#160;</td>
			<td style="width:131px;">SV30010</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">CELLSTAR 6 well plate</td>
			<td style="width:187px;">Greiner Bio-One</td>
			<td style="width:131px;">657 160</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">CELLSTAR 24 well plate</td>
			<td style="width:187px;">Greiner Bio-One</td>
			<td style="width:131px;">662160</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">VWR mini shaker</td>
			<td style="width:187px;">Marshell Sceintific</td>
			<td>12520-956</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Hemocytometer</td>
			<td style="width:187px;">Fisher Sceintific&#160;</td>
			<td>267110</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">FuGENE HD Transfection Reagent</td>
			<td style="width:187px;">Promega</td>
			<td style="width:131px;">E2311</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">MEGAscrip T7 Transcription Kit</td>
			<td style="width:187px;">Ambion</td>
			<td style="width:131px;">AM1334</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:415px;">Quick-RNA MiniPrep (RNA purification Kit)</td>
			<td style="width:187px;">Zymo Research</td>
			<td style="width:131px;">R1054</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">VistaVision Inverted microscope</td>
			<td>VWR</td>
			<td style="width:131px;"></td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:415px;">9MP USB2.0 Microscope Digital Camera + Advanced Software</td>
			<td style="width:187px;">AmScope</td>
			<td style="width:131px;">MU-900</td>
			<td style="width:396px;">Image acquisition using ToupView software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:415px;">PureLink Quick Gel Extraction Kit</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:131px;">K210012</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:415px;">Fetal Bovine Serum</td>
			<td style="width:187px;">GenDepot</td>
			<td style="width:131px;">F0600-050</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Methotrexate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:131px;">A6770-10</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Hygromycin B</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:131px;">HY068-L6</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:415px;">Copper sulphate</td>
			<td style="width:187px;">Macron Fine Chemicals</td>
			<td style="width:131px;">4448-02</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">S2 cells</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:131px;">R69007</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:415px;">S2-SerrateTom cells</td>
			<td style="width:187px;"></td>
			<td style="width:131px;"></td>
			<td style="width:396px;">Gift from R. Fleming (Fleming et al, Development, 2013)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">S2-Delta cells</td>
			<td style="width:187px;">DGRC</td>
			<td style="width:131px;">152</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">S2-Notch cells</td>
			<td style="width:187px;">DGRC</td>
			<td style="width:131px;">154</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">pMT-Delta vector</td>
			<td style="width:187px;">DGRC</td>
			<td style="width:131px;">1021</td>
			<td style="width:396px;">Gift from S. Artavanis-Tsakonas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:415px;">pMT-Serrate vector</td>
			<td style="width:187px;"></td>
			<td style="width:131px;"></td>
			<td style="width:396px;">Gift from Ken Irvine (Okajima et al, JBC, 2003)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">pMT-Notch vector</td>
			<td style="width:187px;">DGRC</td>
			<td style="width:131px;">1022</td>
			<td style="width:396px;">Gift from S. Artavanis-Tsakonas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">pAc5.1-EGFP</td>
			<td style="width:187px;"></td>
			<td style="width:131px;"></td>
			<td style="width:396px;">Gift from Hugo Bellen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">TaqMan RNA-to-Ct 1-Step Kit</td>
			<td style="width:187px;">Applied Biosystem</td>
			<td style="width:131px;">1611091</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">TaqMan Gene Expression Assay for CG9996 (Shams)</td>
			<td style="width:187px;">Applied Biosystem</td>
			<td>Dm02144576_g1&#160;</td>
			<td style="width:396px;">with FAM-MGB dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">TaqMan Gene Expression Assay for CG7939 (RpL32)</td>
			<td style="width:187px;">Applied Biosystem</td>
			<td>Dm02151827_g1</td>
			<td style="width:396px;">with FAM-MGB dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Applied Biosystems 7900HT Fast Real-Time PCR system</td>
			<td style="width:187px;">Applied Biosystem</td>
			<td style="width:131px;">4351405</td>
			<td style="width:396px;">96-well Block module</td>
		</tr>
	</tbody>
</table>

cell aggregation assays to evaluate the binding of the drosophila notch with trans-ligands and its inhibition by cis-ligands

Entalhe de sinalização é um sistema de comunicação célula-célula evolutivamente conservada usado amplamente no desenvolvimento animal e manutenção de adulta. Interação do receptor Notch com ligantes de vizinhas células induz a ativação da via de sinalização (trans-ativação), enquanto a interação com ligantes da mesma célula inibe a sinalização (cis-inibição). Equilíbrio adequado entre trans-ativação e cis-inibição ajuda a estabelecer os níveis ideais de entalhe sinalização em alguns contextos durante o desenvolvimento do animal. Por causa dos domínios de expressão sobreposição de entalhe e seus ligantes em muitos tipos de células e a existência de mecanismos de feedback, estudando os efeitos de uma determinada modificação pós-traducional na trans- contra cis-interações de entalhe e seus ligantes na vivo é difícil. Aqui, descrevemos um protocolo para o uso de células de Drosophila S2 em célula-agregação ensaios para avaliar os efeitos de derrubar um modificador de via Notch na ligação de entalhe para cada ligante trans e cis. Células S2 estàvel ou transitoriamente transfectadas com um vetor de entalhe-expressando são misturadas com células expressando cada ligante de entalhe (S2-Delta ou S2-Serrate). Trans-ligação entre o receptor e ligantes resulta na formação de agregados de células heterotypic e é medido em termos de número de agregados por mL, composto de &#62; 6 células. Para examinar o efeito inibitório do cis-ligantes, S2 células expressando co entalhe e cada ligante são misturadas com células S2-Delta ou S2-Serrate e o número de agregados é quantificado como descrito acima. A diminuição relativa do número de agregados devido à presença de cis-ligantes fornece uma medida de cis-ligand-mediados por inibição da trans-ligação. Estes ensaios simples podem fornecer dados semi quantitativos sobre os efeitos de manipulações genéticas ou farmacológicos na ligação de entalhe para seus ligantes e podem ajudar a decifrar os mecanismos moleculares subjacentes os efeitos na vivo de tais manipulações na sinalização Notch.

As nossas observações na vivo sugeriram que a perda dos resultados xylosyltransferase gene Souza em ganho de entalhe sinalização devido ao aumentado mediada por Delta trans-ativação de entalhe sem afetar o cis-inibição da Entalhe por ligantes8. Para testar esta noção, realizaram-se ensaios de agregação em vitro celular. Primeiro, Souza expressão nas células S2 foi derrubado usando Souza ds...

Watch this Scientific Journal Video about Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands at JoVE.com

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexidade dos sistemas na vivo torna difícil distinguir entre a ativação e a inibição do receptor do entalhe por trans- e cis-ligantes, respectivamente. Aqui, apresentamos um protocolo baseado em vitro ensaios de agregação de célula para avaliação qualitativa e semi-quantitativa da vinculação de entalhe de Drosophila Trans-ligantes vs cis-ligantes.

Celular agregação ensaios para avaliar a vinculação do entalhe drosófila com Trans-ligantes e sua inibição pelo Cis-ligantes

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

cell-aggregation-assays-to-evaluate-binding-drosophila-notch-with

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

7.4K visualizações.  Baylor College of Medicine. Complexidade dos sistemas na vivo torna difícil distinguir entre a ativação e a inibição do receptor do entalhe por trans- e cis-ligantes, respectivamente. Aqui, apresentamos um protocolo baseado em vitro ensaios de agregação de célula para avaliação qualitativa e semi-quantitativa da vinculação de entalhe de Drosophila Trans-ligantes vs cis-ligantes.

Vídeo: Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Produção e Uso de Lentivírus a seletivamente transduzem Primária Oligodendrocyte células precursoras para<em&gt; In Vitro</em&gt; Os ensaios mielinização

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

Biologia do Desenvolvimento

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

Ensaiando populações de células do sangue<em&gt; Drosophila melanogaster</em&gt; Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Desenvolvimento de um<em&gt; In Vitro</em&gt; Ensaio para avaliar contrátil Função de células mesenquimais que a transição epitelial-mesenquimal Sofreu

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Métodos para examinar a linfa Gland e Hemócitos em<em&gt; Drosophila</em&gt; Larvas

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Estimulação da sinalização Notch no mouse osteoclastos Precursores

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Bioengenharia de microrganismos humanizados de medula óssea no mouse e sua visualização por imagem ao vivo

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissecção e imunofluorescência coloração de cogumelo corpo e fotoreceptor neurônios em cérebros adultos Drosophila melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Renderização tridimensional e análise de Immunolabeled, esclareceu humana da placenta vilosa redes vasculares

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

Sondando a célula mecânica com pinça óptica de grânulo-livre no embrião da drosófila

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a G&#945;12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

Um ensaio de baseada em célula para investigar não-músculo Myosin II contratilidade através o dobrado-gastrulação sinalização via em Drosophila S2R + células

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...