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

1. Disección de ovarioles <ol><li> Transferir unos quince 2-4 días de edad, las moscas hembras y unos pocos hombres a un nuevo vial de alimentos mosca con agregado de levadura seca activa. Utilice algodón como un tapón para los viales, y añadir unas gotas de agua para el algodón para mantener la humedad alta. </li><li> Colocar el vial en una incubadora a 25 ° C durante 14-16 horas para maximizar el número de la etapa 8-10 cámaras de huevos. NOTA: El tiempo de incubación varía dependiendo de la temperatura y la fase deseada. </li><li> Preparar los medios de disección, 0,1 M KPO 4 y NP40 soluciones según sea necesario para el día siguiente (ver Materiales). </li><li> Anestesiar las moscas utilizando CO 2, y poner bajo un microscopio de disección. Pipet varias gotas de medios disección en cada cavidad en la diapositiva depresión de vidrio. </li><li> Mantenga fórceps en cada mano en un ángulo de aproximadamente 30 ° a la superficie de la mesa y orientar una mosca hembra con las alas hacia abajo. Con la mano no dominante, recoger la hembra usando pinzas, agarrándola de la unanterior del abdomen, cerca del tórax, y sumerja en el medio de su disección en la diapositiva depresión (Figura 1A recuadro). </li><li> Con el otro par de pinzas, sujete el exoesqueleto en la ventral posterior del abdomen y la traspasará. Coge los ovarios en el extremo posterior, a continuación, tire hacia fuera del abdomen y el lugar en el medio nuevo en el otro lado de la corredera de la depresión. (Figura 1A recuadro). Evite tirar aparte el intestino. Deseche la canal. </li><li> Repita con el resto de las moscas hembras hasta que se disecan los ovarios. Deseche los varones. </li><li> Con un par de pinzas, mantenga un ovario en el extremo posterior (cerca de las grandes cámaras de huevos), y con la otra mano usar un par de pinzas para pellizcar una de las cámaras de huevos más anteriores (germarium). (Figura 1A). </li><li> Poco a poco y de manera constante, tirar de una cadena ovariole fuera de la vaina ovario. Continuar a diseccionar ovarioles individualmente hasta que todas las cámaras de huevo deseados son removed. </li><li> Repita los pasos 1.7 a 1.8 para todos los ovarios disecados. </li><li> Una vez completa, traslado ovarioles a un tubo de 0,6 ml con una pipeta de transferencia de plástico. </li><li> Deje ovarioles depositan en el fondo del tubo, a continuación, proceder a los pasos de fijación y tinción. </li></ol> 2. Inmunofluorescencia (IF) La tinción de ovarioles <ol><li> Retire con cuidado los medios disección de ovarioles con una pipeta. Asegúrese de no eliminar cualquier ovarioles o cámaras de huevo. Deje aproximadamente 50 l de ovarioles y medios de disección. </li><li> Añadir 50 l de 16% de paraformaldehído a 150 ml de 0,1 M KPO4 de búfer en un tubo, y añadir la solución de cámaras de huevos para solucionarlos. Incubar mientras se balancea durante 10 min. PRECAUCIÓN: paraformaldehído es tóxico. </li><li> Retire el fijador y enjuague dos veces con tampón de lavado NP40 0,5 ml. </li><li> Lavar dos veces durante 15 min cada uno a temperatura ambiente. </li><li> Consulte la norma SI protocolo 15 para los pasos posteriores. <ol><li> Brevemente, después de la fijación, añadiranticuerpos primarios a las diluciones apropiadas y se incuban O / N a 4 ° C. Lave en NP40 varias veces, a continuación, añadir anticuerpos secundarios (1: 200 diluciones). </li><li> Lavar varias veces en NP40, a continuación, añadir DAPI (1: 1000) durante 10 min, a continuación, repetir los lavados. Una vez SI protocolo es completa, añadir 200 l de glicerol al 50% en PBS para cámaras de huevos y dejar reposar a temperatura ambiente durante 2 horas. </li><li> Retire la solución, reemplazarlo con un 70% de glicerol en PBS, y cubrir con papel aluminio. Coloque la muestra en 4 ᵒC hasta que esté listo para el montaje. Mientras tanto, continúe con el paso 3. </li></ol></li></ol> 3. Preparación previa de Glicerina Jalea Slides <ol><li> Medir al menos 4 ml de glicerina jalea usando una espátula y llenar un tubo de cultivo de célula de vidrio hasta la marca de medio camino. Derretir la jalea de glicerina en un baño de agua a 55 ° C durante 30 min. </li><li> Verter una pequeña gota de glicerol, aproximadamente 300 l, a partir de la solución madre en dos portaobjetos de microscopio. El uso de un tejido, extendido glicerol en una delgada layer en cada una de las diapositivas. Esto proporciona una base y permite una fácil extracción de gelatina de glicerina en el paso 5.6. </li><li> Corte la punta de una punta de pipeta de 1.000 l filtrada. El uso de puntas de pipeta de corte para transferir 1.000-1.500 l de jalea caliente glicerina lentamente a cada portaobjetos. Tenga cuidado para evitar burbujas. </li><li> Deje diapositivas jalea glicerina sientan durante 5 minutos a temperatura ambiente para ajustar. </li><li> Coloque cada diapositiva jalea glicerina en 60 mm × 15 mm plato de Petri y cubrir con papel plástico. Lugar a 4 ° CO / N. Opcionalmente, tienda de glicerina diapositivas jalea de hasta 5 días a 4 ° C. </li></ol> 4. Montaje de cámaras de huevos <ol><li> Usando muestras de la etapa 2.5, pipeta varias gotas 3 l de cámaras de huevos en 70% de glicerol a través de uno de glicerina jalea diapositiva. Continuar pipeteo 3 mu l gotas hasta que todas las cámaras de huevos están en esta diapositiva. Asegúrese de que cada gota contiene al menos diez cámaras de huevos. Mientras tanto, calentar un tubo de cultivo de gelatina de glicerina en un 55 ° C baño de agua. </li><li> Colocar el portaobjetos jalea glicerina con cámaras de huevos bajo un microscopio de disección. Ajustar la ampliación de modo que sólo una gota de cámaras de huevo está en el campo de visión (Figura 1B). </li><li> El uso de una aguja de calibre 30 como una cuchara, coger la cámara de la etapa de huevo deseado. Cuando sea necesario, cámaras de huevos deseados separados de una cadena ovariole utilizando la aguja como un cuchillo. </li><li> Para evitar los desechos que puedan interferir con la imagen, transferir la cámara de huevos a la segunda diapositiva jalea glicerina, con la anterior frente a la izquierda (Figura 1E -1). Repita hasta que hay por lo menos siete cámaras de huevos alineados en una columna (Figura 1C). </li><li> Formar nuevas columnas de siete hasta que todas las cámaras de huevos deseados se transfieren a la segunda diapositiva jalea glicerina. </li><li> Corte un pequeño trozo de tejido y giro. Use esto para absorber el exceso de PBS-glicerol a partir de las cámaras de huevos tocando ligeramente cada una. Tenga cuidado de no eliminar cámaras de huevos. </li><li> Cortar lapunta de una punta de pipeta 200 l filtrada. Utilice esta transferir 150 l de glicerina jalea para cubrir todas las columnas de cámaras de huevos. NOTA: La cantidad de glicerina jalea necesario para cubrir las columnas se basa en las etapas de la cámara de huevo y el número de columnas. La cantidad puede ser ajustada. </li><li> Deje cámaras de huevos montados sientan durante 5 minutos a temperatura ambiente hasta que la capa superior de la jalea de la glicerina se solidifica por completo (Figura 1D). </li><li> Ponga los portaobjetos de cámaras de huevos montados en una placa de 60 mm x 15 mm de Petri y cubrir con papel plástico. Almacenar a 4 ° CO / N para permitir que las capas de gelatina glicerina para solidifican juntos (lugar en la oscuridad si existe preocupación por photobleaching). </li></ol> 5. Preparación de Cámaras de huevo de la Imagen <ol><li> Coloque la diapositiva de cámaras de huevos montados bajo un microscopio de disección. Ajustar la ampliación de modo que sólo una columna de cámaras de huevo está en el campo de visión. </li><li> Con un bisturí miniatura ángulo de 45 °, corte un straight línea a lo largo del lado derecho de la primera columna de cámaras de huevos (cerca de la parte posterior de las cámaras de huevos). (Figura 1D-E) </li><li> A continuación, cortar por encima de la primera cámara de huevos en la parte superior y por debajo de la última cámara de huevos en la columna. En este punto la columna de cámaras de huevos se debe cortar en tres lados. </li><li> El uso de un microknife, rebanada a lo largo del lado izquierdo de la columna, lo más cerca posible a la cara anterior de las cámaras de huevo (Figura 1E) -3. </li><li> Pipetear 100 l de frío 1X PBT sobre la columna de la corte. </li><li> Use una espátula de bordes redondeados para eliminar el exceso de gelatina de glicerina en torno a tres lados de la columna de la corte de cámaras de huevos y descartar, dejando el resto de la diapositiva. </li><li> Inserte la espátula en el corte hecho en el lado anterior de cámaras de huevos y separar la columna desde el bloque de gelatina de glicerina primaria. </li><li> Preparar las muestras, ya sea para invertidas (5.8.1) o vertical (5.8.2) de microscopía. <ol><li> Para Inverted Microscope: t Transferenciaque la columna de cámaras de huevos a un cubreobjetos con cara anterior de cámaras de huevos hacia abajo. Repita los pasos 5.2 a 5.8 hasta que se hayan eliminado todas las columnas de la jalea de glicerina y montado. Añadir un par de gotas de 50% PBS-glicerol en cada bloque de cámaras de huevos para evitar que se sequen. Cámaras de huevos imagen directamente sobre cubreobjetos. </li><li> Para microscopio vertical: Transferir la columna de cámaras de huevos a un portaobjetos de microscopio con la cara anterior hacia arriba. Repita los pasos 5.2 a 5.9 hasta que se hayan eliminado todas las columnas de la jalea de glicerina y montadas en portaobjetos de microscopio. Añadir un par de gotas de 50% PBS-glicerol en cada bloque de cámaras de huevos y los bloques de la cubierta con un cubreobjetos ½ # 1. </li></ol></li></ol>

<ol>
	<li>Hudson, A. M., Cooley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+oogenesis.">Methods for studying oogenesis.</a> Methods. 68 (1), 207-217 (2014).</li><li>Bastock, R., St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+oogenesis.">Drosophila oogenesis.</a> Curr Biol. 18 (23), R1082-R1087 (2008).</li><li>Wu, X., Tanwar, P. S., Raftery, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+follicle+cells:+morphogenesis+in+an+eggshell.">Drosophila follicle cells: morphogenesis in an eggshell.</a> Semin Cell Dev Biol. 19 (3), 271-282 (2008).</li><li>Bilder, D., Haigo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+morphogenetic+repertoire:+perspectives+from+the+Drosophila+egg.">Expanding the morphogenetic repertoire: perspectives from the Drosophila egg.</a> Dev Cell. 22 (1), 12-23 (2012).</li><li>Montell, D. J., Yoon, W. H., Starz-Gaiano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group+choreography:+mechanisms+orchestrating+the+collective+movement+of+border+cells.">Group choreography: mechanisms orchestrating the collective movement of border cells.</a> Nat Rev Mol Cell Biol. 13 (10), 631-645 (2012).</li><li>Spradling, A. C., Bate, M., Marinez-Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+oogenesis.">Developmental genetics of oogenesis.</a> The Development of Drosophila melanogaster. , 1-70 (1993).</li><li>Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Border-cell+migration:+the+race+is+on.">Border-cell migration: the race is on.</a> Nat Rev Mol Cell Biol. 4 (1), 13-24 (2003).</li><li>Silver, D., Geisbrecht, E., Montell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+JAK/STAT+signaling+throughout+border+cell+migration+in+Drosophila.">Requirement for JAK/STAT signaling throughout border cell migration in Drosophila.</a> Development. 132 (15), 3483-3492 (2005).</li><li>Ghiglione, C., 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=The+Drosophila+cytokine+receptor+Domeless+controls+border+cell+migration+and+epithelial+polarization+during+oogenesis.">The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis.</a> Development. 129 (23), 5437-5447 (2002).</li><li>Denef, N., Sch&#252;pbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning:+JAK-STAT+signalling+in+the+Drosophila+follicular+epithelium.">Patterning: JAK-STAT signalling in the Drosophila follicular epithelium.</a> Curr Biol. 13 (10), R388-R390 (2003).</li><li>Beccari, S., Teixeira, L., R&#248;rth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+JAK/STAT+pathway+is+required+for+border+cell+migration+during+Drosophila+oogenesis.">The JAK/STAT pathway is required for border cell migration during Drosophila oogenesis.</a> Mech Dev. 111 (1-2), 115-123 (2002).</li><li>Montell, D., Rorth, P., Spradling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=slow+border+cells,+a+locus+required+for+a+developmentally+regulated+cell+migration+during+oogenesis,+encodes+Drosophila+C/EBP.">slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP.</a> Cell. 71 (1), 51-62 (1992).</li><li>Xi, R., McGregor, J., Harrison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gradient+of+JAK+pathway+activity+patterns+the+anterior-posterior+axis+of+the+follicular+epithelium.">A gradient of JAK pathway activity patterns the anterior-posterior axis of the follicular epithelium.</a> Dev Cell. 4 (2), 167-177 (2003).</li><li>Toomre, D., Bewersdorf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+wave+of+cellular+imaging.">A new wave of cellular imaging.</a> Annu Rev Cell Dev Biol. 26, 285-314 (2010).</li><li>McDonald, J., Pinheiro, E., Kadlec, L., Schupbach, T., Montell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+EGFR+ligands+participate+in+guiding+migrating+border+cells.">Multiple EGFR ligands participate in guiding migrating border cells.</a> Dev Biol. 296 (1), 94-103 (2006).</li><li>Van de Bor, V., Zimniak, G., C&#233;r&#233;zo, D., Schaub, S., Noselli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+localisation+of+cytokine+mRNA+is+essential+for+JAK/STAT+activation+during+cell+invasiveness.">Asymmetric localisation of cytokine mRNA is essential for JAK/STAT activation during cell invasiveness.</a> Development. 138 (7), 1383-1393 (2011).</li><li>Hayashi, Y., 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=Glypicans+regulate+JAK/STAT+signaling+and+distribution+of+the+Unpaired+morphogen.">Glypicans regulate JAK/STAT signaling and distribution of the Unpaired morphogen.</a> Development. 139 (22), 4162-4171 (2012).</li><li>Airoldi, S. J., McLean, P. F., Shimada, Y., Cooley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+protein+movement+in+syncytial+Drosophila+follicle+cells.">Intercellular protein movement in syncytial Drosophila follicle cells.</a> J Cell Sci. 124 (Pt 23), 4077-4086 (2011).</li><li>R&#248;rth, P., 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=Systematic+gain-of-function+genetics+in+Drosophila).">Systematic gain-of-function genetics in Drosophila).</a> Development. 125 (6), 1049-1057 (1998).</li><li>Lee, T., Lee, A., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Drosophila+mushroom+bodies:+sequential+generation+of+three+distinct+types+of+neurons+from+a+neuroblast.">Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.</a> Development. 126 (18), 4065-4076 (1999).</li><li>Shimada, Y., Burn, K. M., Niwa, R., Cooley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+response+of+protein+localization+and+microtubule+organization+to+nutrient+stress+during+Drosophila+early+oogenesis.">Reversible response of protein localization and microtubule organization to nutrient stress during Drosophila early oogenesis.</a> Dev Biol. 355 (2), 250-262 (2011).</li><li>Qui&#241;ones-Coello, A. T., 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=Exploring+strategies+for+protein+trapping+in+Drosophila.">Exploring strategies for protein trapping in Drosophila.</a> Genetics. 175 (3), 1089-1104 (2007).</li><li>Buszczak, 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=The+carnegie+protein+trap+library:+a+versatile+tool+for+Drosophila+developmental+studies.">The carnegie protein trap library: a versatile tool for Drosophila developmental studies.</a> Genetics. 175 (3), 1505-1531 (2007).</li><li>Belu, 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=Upright+imaging+of+Drosophila+embryos.">Upright imaging of Drosophila embryos.</a> J Vis Exp. (43), (2010).</li><li>Witzberger, M. M., Fitzpatrick, J. A., Crowley, J. C., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=End-on+imaging:+a+new+perspective+on+dorsoventral+development+in+Drosophila+embryos.">End-on imaging: a new perspective on dorsoventral development in Drosophila embryos.</a> Dev Dyn. 237 (11), 3252-3259 (2008).</li></ol>

The authors have no competing financial interests.

Aquí se describe un método para montar y la imagen pequeña, el desarrollo de cámaras de huevos desde una perspectiva final sobre. Las técnicas comunes para cámaras de huevos de imagen se optimizan para las vistas laterales y permiten principalmente visualización precisa de las células del folículo medio-lateral cuando se tiñen con anticuerpos fluorescentes. El uso de la Z-pilas o 3-D ayudas reconstrucciones en la visualización de múltiples planos focales, pero sigue siendo insuficiente para la resolución su...

Estudio de Drosophila melanogaster ha proporcionado grandes conocimientos sobre la regulación genética de una amplia gama de fenómenos. En particular, ha habido una extensa investigación en el desarrollo de huevo, porque la ovogénesis proporciona una forma manejable para investigar muchos procesos de desarrollo diferentes, incluyendo patrones de tejido, los cambios de polaridad celular, la conmutación del ciclo celular, y la regulación de traducción 1,2,3,4. Un evento morfogénica importante durante la ovogénesis es la especificación, la adquisición de la motilidad, y la migración de un conjunto de células llamadas células de frontera (revisado en 5). Puesto que la migración celular es una característica clave de la morfogénesis animal, y porque la regulación genética de este proceso está bien conservada, mecanismos determinados en las moscas son propensos a ser importante en otros contextos. Por lo tanto, estamos investigando el control molecular de la migración celular frontera. Para nuestros estudios, hemos desarrollado un nuevo método para observar los polos anteriores de desarrollo de los huevos,donde surgen las células de frontera, para examinar cómo se desarrollan en detalle. Dentro del ovario, existen cámaras de huevos en las distintas etapas de desarrollo a lo largo de las cadenas de llamadas ovarioles, que están provistas de un revestimiento delgado (Figura 1). Cada cámara de huevos pasará a formar un huevo. Durante la ovogénesis, varios tipos diferentes de células deben desarrollar de manera coordinada. El D. cámara de huevos melanogaster consta de 16 células de la línea germinal, entre ellos uno de los ovocitos, rodeado de una sola capa de epitelio folicular 6. Un pequeño número de células especializadas, llamadas células polares, surgen en los anteriores y posteriores polos del epitelio. Las células del borde 6-8 se originan en el epitelio anterior de la cámara de huevos, inducida por las células polares 5,7. A mediados de la ovogénesis (etapa 9), las células se desprenden de la frontera de sus vecinos y migran entre las células de la enfermera para alcanzar el ovocito en la parte posterior de la cámara de huevos 5,7. Este movimiento debe ser realizadomientras que las células permanecen en la frontera de un clúster que rodea dos células polares no móviles, haciendo de este un tipo de migración celular colectiva. Migración exitosa del grupo de células frontera garantiza el desarrollo adecuado de la micropilo de la cáscara del huevo, que es necesaria para la fertilización. Las células polares anteriores instruyen destino celular frontera mediante la activación de una cascada de transducción de señales. Célula polar secretan una citoquina, Unpaired (UPD), que se une a un receptor transmembrana, domeless (DOMO), en células del folículo vecino durante la etapa 8 del desarrollo de los ovocitos 8,9. La unión de UPD causa de la tirosina quinasa Janus (JAK) para fosforilan el transductor de señales y activador de la transcripción (STAT) 8,10,11. STAT se mueve entonces al núcleo para activar la transcripción. Células del borde lento (SLBO) es un factor de transcripción que es una diana transcripcional directa de STAT y también se requiere para la migración de células frontera 12. Vistas laterales de cámaras de huevos indican tactividad STAT sombrero está regulada en un gradiente a través del epitelio anterior 8,11,13. Células del folículo más cercanos a las células polares tienen los más altos niveles de STAT activado, por lo que se convierten en células de frontera e invaden el tejido de la línea germinal adyacente. Para entender cómo se especifican las células del borde interior y se desprenden del epitelio, tenemos que observar cómo se organiza el tejido. Si vemos cámaras de huevos desde una perspectiva anterior-en, esperaríamos la simetría radial de la actividad STAT en las células foliculares que rodean las células polares. Una vista frontal también mostrar con mayor precisión las diferencias de las proteínas de membrana y las interfaces de célula-célula antes y durante el desprendimiento de la comparación de células en diferentes planos focales. Porque cámaras de huevos son oblongas y unido entre sí por las células tallo, se instalan en portaobjetos lateralmente, por lo que es difícil de observar la arquitectura anterior. Por lo tanto, toda la información acerca de las células en los polos de la cámara de huevos tieneha inferido a partir de vistas laterales. Aunque alguna información puede ser obtenida a través de algoritmos reconstrucciones 3-D de las secciones ópticas, dispersión de la luz, fotoblanqueo y límites más pobres de la resolución en el eje Z que esta información menos detallada y fiable en ausencia de técnicas costosas como super-resolución microscopía 14 . Otros tipos de formación de imágenes basada en sección (por ejemplo, microscopía electrónica o seccionamiento microtomo) requieren una extensa manipulación de los tejidos, incluyendo la deshidratación, aumentando la probabilidad de artefactos. Por lo tanto, hemos desarrollado un nuevo método para la imagen D. cámaras de huevos melanogaster mientras que en posición vertical. Este método ya ha demostrado su utilidad en la aclaración de cómo están destinados células móviles (ver resultados representativos y Manning et al, en revisión), y es probable que sea más ampliamente valiosa en el estudio de otros aspectos de la ovogénesis.

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			<td height="40" style="height:40px;width:348px;">Name of Reagent/ Equipment</td>
			<td style="width:279px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:283px;">Comments/Description</td>
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		<tr height="96">
			<td height="96" style="height:96px;width:348px;">0.1 M Potassium phosphate Buffer (KPO4 Buffer)</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">Add 3.1 g of NaH2PO4&#8226;H2O and 10.9 g of Na2HPO4 (anhydrous) to distilled H2O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 mo at 4&#176;C.</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:348px;">16% Paraformaldehyde aqueous solution, EM grade</td>
			<td style="width:279px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>methanol-free to preserve GFP fluorescence</td>
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			<td height="20" style="height:20px;width:348px;">30G x 1/2 inch needle&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">BD305106</td>
			<td>Regular bevel</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:348px;">Active Dry Yeast</td>
			<td style="width:279px;">Genesee Scientific</td>
			<td>62-103</td>
			<td>To fatten female flies</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Bovine Serum Albumin</td>
			<td style="width:279px;">PAA-cell culture company</td>
			<td style="width:119px;">A15-701</td>
			<td>Used in NP40 Wash Buffer</td>
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		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Dumont #5 Forceps</td>
			<td style="width:279px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td style="width:283px;">Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td>
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		<tr height="39">
			<td height="39" style="height:39px;width:348px;">DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td style="width:283px;">5mg/ml stock solution&#160;&#160;</td>
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		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;">16140-071</td>
			<td style="width:283px;">Added to supplement dissection medium (10%)&#160;</td>
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			<td height="24" style="height:24px;width:348px;">Glass culture tube</td>
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			<td>47729-576</td>
			<td>14mL</td>
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			<td height="20" style="height:20px;width:348px;">Glass Depression Slides</td>
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			<td>470019-020</td>
			<td>1.2 mm Thick, Double Cavity</td>
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			<td style="width:119px;">IB15760</td>
			<td>70% in PBS</td>
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			<td height="39" style="height:39px;width:348px;">IGEPAL CA-630</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">I3021-500ML</td>
			<td style="width:283px;">(interchangable for Nonidet P-40)&#160; Used in NP40 Wash Buffer&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica Fluorescent Stereoscope&#160;</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica SP5 Confocal Microscope</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td>40x/0.55NA dry objective&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Micro spatula&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">82027-518</td>
			<td>Stainless steel</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microknife&#160;</td>
			<td style="width:279px;">Roboz Surgical Instrument Company</td>
			<td style="width:119px;">37-7546</td>
			<td>45&#7506; angle</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microscope Slide</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">16004-368</td>
			<td>75x25x1 mm</td>
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		<tr height="58">
			<td height="58" style="height:58px;width:348px;">NP40 Wash Buffer</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">50mM TRIS-HCL, 150mM NaCl, 0.5% Ipegal, 1mg/ml BSA, and 0.02% sodium azide</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Penicillin-Streptomycin-Glutamine</td>
			<td style="width:279px;">Life Technologies</td>
			<td>10378-016</td>
			<td>Added to supplement dissection medium (0.6X)</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Petridish- Polysterine, sterile</td>
			<td style="width:279px;">VWR</td>
			<td>82050-548</td>
			<td>60Wx15H mm</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Phosphate Buffer Saline</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3813-10PAK</td>
			<td>10 packs of Powder</td>
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			<td height="20" style="height:20px;width:348px;">Potassium phosphate dibasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3786-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Potassium phosphate monobasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P9791-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Schneider&#8217;s Insect Medium</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;"> 
			11720018</td>
			<td>With L-glutamine and sodium bicarbonate</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:348px;">Sodium azide</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S2002-25G</td>
			<td>Used in fluorescent antibody staining</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Sodium chloride</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S3014-500G</td>
			<td>Used in NP40 Wash Buffer</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">TRIS-HCL</td>
			<td style="width:279px;">IBI Scientific</td>
			<td style="width:119px;">IB70144</td>
			<td>1M TRIS-HCL, pH 7.4</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Volocity 3D Image Analysis Software</td>
			<td style="width:279px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>For processing confocal Z-stacks</td>
		</tr>
	</tbody>
</table>

upright imaging of drosophila egg chambers

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

El método de formación de imágenes en posición vertical nos permitió ver directamente la organización de células en el epitelio folicular anterior en la etapa 8. Un marcador general de destino de las células del folículo, la ausencia Eyes (EYA) de proteína, así como el marcador de ADN nuclear DAPI, mostró incluso expresión a través de este campo de las células, y demostraron que todas las células se podían ver con intensidades de tinción similares (Figura 2B &quot;). Proteínas regulad...

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

Upright Imaging of Drosophila Egg Chambers

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

Imaging Upright de<em&gt; Drosophila</em&gt; Cámaras de huevo

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

upright-imaging-of-drosophila-egg-chambers

Research

JoVE Journal

Developmental Biology

Upright Imaging of Drosophila Egg Chambers

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

Video: Upright Imaging of Drosophila Egg Chambers

Live-imaging of the Drosophila Pupal Eye

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

Live-imaging of the Drosophila Pupal Eye

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

Live-proyección de imagen de la<em&gt; Drosophila</em&gt; Pupal Ojo

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

Investigación

Biología del Desarrollo

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Usando<em&gt; Ex Vivo</em&gt; Culturas Upright Gotita de enteros fetales Órganos para estudiar los procesos de desarrollo durante Ratón Organogénesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Imaging Calcium in Drosophila at Egg Activation

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

Imaging Calcium in Drosophila at Egg Activation

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

El calcio de imágenes en<em&gt; Drosophila</em&gt; En la activación del huevo

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

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

A largo plazo de imágenes en vivo<em&gt; Drosophila</em&gt; Disco de ojos

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

En Vivo Proyección de imagen del músculo-tendón morfogénesis en Drosophila pupa

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Análisis de la dinámica de Actomyosin en escalas celulares y de tejidos locales utilizando películas de lapso de tiempo de cámaras de huevo de Drosophila cultivada

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

Imagen en vivo y análisis de contracciones musculares en Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

Descongelación, cultivo y criopreservación de líneas de células de Drosophila

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Human Egg Maturity Assessment and Its Clinical Application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

Evaluación de la madurez del óvulo humano y su aplicación clínica

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...

Imaging Dpp Release from a Drosophila Wing Disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-&#946; superfamily includes TGF-&#946;, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-&#946; superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Imaging Dpp Release from a Drosophila Wing Disc

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.