Name: dissection and staining of drosophila pupal ovaries
Uploaded: 2018-03-02T14:30:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Dissection and Staining of Drosophila Pupal Ovaries at JoVE.com

Esta investigación fue apoyada por los institutos nacionales de salud (to1 GM079351 a D.K.). Damos las gracias por sus consejos sobre disecciones de ovario pupa basados en su Protocolo original Dorothea Godt. También agradecemos a Amy Reilein por su ayuda y comentarios sobre el manuscrito.

1. oviposición
<ol><li>Combinar unos diez hombres y quince mujeres Drosophila moscas del genotipo deseado en un frasco de alimento mosca rico normal con levadura. Para evitar hacinamiento el frasco, deje que acoplado las hembras a desovar durante no más de 2 – 4 h14.</li>
	<li>Transferencia de los adultos del frasco en un frasco nuevo golpeando el frasco apertura contra un frasco diferente con la comida de mosca. Permita que los huevos se convierten en larvas a temperatura ambiente durante 3-4 días.</li>
</ol>2. seleccionar las larvas hembra
<ol><li>Usando un pincel fino húmedo con cerdas suaves, hacer un movimiento del balanceo con el cepillo a lo largo de la pared de la cubeta de transferencia de larvas de tercer estadio errantes del frasco a un vaso bien lleno hasta el borde con 1 x solución salina tamponada con fosfato (PBS). Lavar los restos de alimento de las larvas a transferirlos a otro bien con PBS 1 x.</li>
	<li>Separar las larvas machos y hembras con unas pinzas. Identificar las larvas masculinas por un par de testículos grandes, redondos y transparente incrustado en la grasa corporal en el lado lateral del cuerpo aproximadamente dos tercios hacia abajo desde su extremo anterior15. Larvas hembra son en promedio más grandes, menos transparente que los machos y tienen gónadas mucho más pequeñas que son más difíciles de detectar.</li>
	<li>Seleccione contra larvas macho para recoger las larvas hembra. Recoger las hembras por lo menos diez del pozo. Coloque las larvas hembra en un separado bien llenado de PBS 1 x.</li>
	<li>Uso de fórceps para transferir las larvas hembra suavemente en un frasco nuevo de alimentos frescos que mosca complementan con levadura.</li>
	<li>Coloque el frasco con las larvas hembra en un lugar oscuro para facilitar pupariación16. Durante todo el día, controlar las larvas de pupariación. Cada larva se inmoviliza y se convierte en una prepupa, círculo la prepupa contra el vial y registre el tiempo aproximado cuando forma en una prepupa. Identificar prepupas por la protrusión de espiráculos anteriores y momento de formación del pupario17. Permiten las prepupas a desarrollar hasta el punto deseado de tiempo (medido en horas después de la formación del pupario, APF). Nota: Cualquier animal que se someten a la formación del pupario dentro de un intervalo h aproximadamente 10 puede ser considerado una prepupa. El estadio pupal comienza aproximadamente 12 horas después de la formación del pupario después de una muda interior.</li>
</ol>3. preparación de pupas para tinción de anticuerpos
<ol><li>
Formar una pipeta de vidrio delgado para ser utilizado en pasos posteriores para limpiar el cuerpo de grasa pupal.
	<ol>
<li>Derretir la punta del cristal de una pipeta Pasteur sobre un mechero de Bunsen. Como se derrite el vidrio, utilice pinzas para tirar la punta horizontalmente lejos del resto de la pipeta para formar una punta más fina.</li>
		<li>Después de enfriar, romper una pequeña porción de la punta de la pipeta para formar una abertura circular limpia. Coloque un bulbo al otro extremo de la pipeta.</li>
		<li>Cargar la pipeta con PBS 1 x.</li>
	</ol>
</li>
	<li>Para cosechar las pupas, que se pegan a la pared del frasco, aplique una pequeña gota de agua a lo largo de la zona de contacto entre pupa y vial. Esperar 1-2 minutos para permitir que la proteína disolver pegamento antes de levantar suavemente la pupa de la pared con un pincel fino húmedo. Traslado de la pupa a un vaso bien llenada de 1 x PBS. Dedicar un solo pozo por pupa para evitar hacinamiento el pozo.</li>
	<li>Mientras agarra el extremo posterior de la pupa con un par de pinzas, romper cuidadosamente la porción anterior de la envoltura pupal con otro par hasta que la cabeza de la pupa.</li>
	<li>Sujete la punta más anterior de la cabeza de pupa con pinzas y tire suavemente de la pupa de su envoltura pupal. Nota: Una porción de la grasa corporal puede derramarse durante este proceso.</li>
	<li>Separar y desechar la mitad anterior de la pupa de la mitad posterior hasta que quede solamente el saco abdominal.</li>
	<li>
Extracto de las células de grasa corporal del saco abdominal.
	<ol>
<li>Agarrar el saco abdominal contra el fondo del pozo con pinzas en una mano mientras sostiene la pipeta de vidrio delgado llenada de 1 x PBS en otro lado.</li>
		<li>Apunte la punta de la pipeta fina hacia la apertura de la bolsa y lentamente pipetee 1 x PBS en el abdomen para lavar las células de grasa corporal que rodean los ovarios pupas. Nota: Los ovarios son un par de estructuras pequeñas, translúcidas, estriados y oblongos que debe permanecer dentro del saco abdominal.</li>
		<li>Lavar que las células de la grasa corporal hasta por lo menos dos tercios de la grasa corporal se ha ido o hasta que los ovarios son visibles cerca de la abertura de la bolsa abdominal. Es muy importante realizar este paso poco a poco para que no se lave los ovarios en el abdomen por accidente.</li>
	</ol>
</li>
	<li>Examinar el pozo para comprobar si los ovarios han derramado hacia fuera durante el lavado de células de grasa corporal. Si los ovarios no son visibles dentro del pozo, se han mantenido en el abdomen. Si los ovarios han salido, coloque una pupa nuevo en el pozo y repita este paso.</li>
	<li>Transferir el abdomen en una cámara transparente cubreobjetos llenada de tampón de fijación (1 x PBS paraformaldehído al 4%) y coloque la tapa en la parte superior. Para asegurar un número suficiente de ovarios soportar la coloración y el proceso de montaje, diseccionar los ovarios más que lo necesario. PRECAUCIÓN: El búfer de fijación contiene paraformaldehído, que es tóxico. Por favor, use protección adecuada como guantes, gafas de seguridad, etcetera.</li>
</ol>4. inmunohistoquímica
<ol><li>Durante el proceso de tinción, utilice pinzas para Presione suavemente hacia abajo cualquier ovarios flotantes hasta el fondo del pozo de cristal para asegurarse de que los ovarios están completamente inmersos en solución de anticuerpo.</li>
	<li>Tiras de cinta de doble cara sobre la superficie plana de un Nutator y luego coloque el cubreobjetos "chambré" en la superficie adhesiva.</li>
	<li>Incubar los ovarios en buffer de fijación del paso 3.7 durante 15 min a temperatura ambiente.</li>
	<li>Enjuague los ovarios tres veces en PBS 1 x con 1% Tritón X-100 para 5, 10 y 45 minutos (1 h en total), respectivamente, para permitir la permeabilización completa.</li>
	<li>Bloquear los ovarios en el 10% de suero normal de cabra de 1 x PBS con 0,5% Tritón X-100 durante 30 minutos.</li>
	<li>Incubar durante una noche los ovarios en 600 μl de anticuerpo primario de elección diluido a la concentración apropiada en PBS 1 x con 0.5% Tritón X-100 a 4 ° C.</li>
	<li>Enjuague los ovarios tres veces durante 5 minutos en PBS 1 x con 0.5% Tritón X-100.</li>
	<li>Incubar los ovarios para 2 h en 600 μl del anticuerpo secundario de elección había diluido a la concentración apropiada en PBS 1 x con 0.5% Tritón X-100 a temperatura ambiente.</li>
	<li>Enjuague los ovarios dos veces durante 5 minutos en PBS 1 x con 0,5% Tritón X-100 y una vez durante 5 minutos en PBS 1 x.</li>
</ol>5. disección y montaje pupas ovarios
<ol><li>Coloque una gota pequeña de 1 x PBS sobre un portaobjetos de microscopio. Uso de fórceps para transferir el saco abdominal desde el cubre-objetos de cámaras a un vaso bien llenan de PBS 1 x. Dedicar un solo pozo por pupa para evitar hacinamiento el pozo.</li>
	<li>Desgarrando con fórceps el saco abdominal y cualquier resto grasa corporal. Nota: Los ovarios, que son un par de estructuras pequeñas, translúcidas, estriados y oblongos, deben verse dentro del pozo. Los ovarios son a menudo fuertemente rodeados por la grasa corporal, por lo que es importante para asegurarse de que todo el cuerpo de grasa es completamente rasgada aparte.</li>
	<li>Transferir los ovarios desde el pozo a la gota de 1 x PBS en el microscopio diapositiva sujetando el centro de los ovarios entre las puntas de las pinzas. Es muy importante usar un agarre firme sin exprimir los ovarios para no perder o destruir durante la transferencia. Añadir unas gotas de 1 x PBS a la corredera cuando la solución se seca con el tiempo.</li>
	<li>Una vez los ovarios han sido disecados y transferido a lo portaobjetos, pipeta 40 μl de medio de montaje sobre un cubreobjetos de 22 x 22 mm y el cubreobjetos con cuidado encima de los ovarios. Deje que la corredera seque durante la noche antes de la proyección de imagen.</li>
</ol>

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Los autores no tienen conflictos de intereses.

El paso más crítico y difícil de este protocolo consiste en la preparación de ovarios pupas antes de la fijación. Para asegurarse de que los ovarios, pequeño y enterrados por las células de grasa corporal dentro de la pupa del abdomen, se tiñen suficientemente con los anticuerpos, es importante no sólo romper una abertura grande en el saco abdominal con pinzas, pero también extraer las células del cuerpo de grasa que obstruyen la ovarios de los anticuerpos. Ejecución exitosa de este paso requiere aplicación ...

Investigación de células madre usando Drosophila ovarios ha expandido ampliamente desde la primera documentación de la célula de vástago hueco1,2,3,4. Tras el desarrollo de herramientas genéticas del seguimiento del linaje, ovario del Drosophila disecciones se han utilizado comúnmente para el estudio de linajes de células madre y señalización vías que regulan el mantenimiento de células madre, proliferación y suerte en el nicho de células madre. Conocimiento de estas vías de señalización puede dar ideas sobre posibles causas de los cánceres que se originan en la célula de vástago aberrante actividad5,6,7. Recientemente se ha demostrado que células madre somáticas en el ovario del Drosophila , conocidas como células de vástago del folículo (Eve), fuertemente se asemejan a mamífero las células intestinales de la madre en muchos aspectos de su organización8. Por esta razón, los ovarios de la Drosophila son un sistema modelo muy útil para estudiar el comportamiento de la célula de vástago.
Mientras que los ovarios de larvarios y adultos ofrecen pistas al desarrollo temprano de la célula de vástago y organización de la célula de vástago final en el nicho, respectivamente, el ovario pupa es una estructura intermedia en la que las células somáticas y germinales reorganizar y establecer sus identidades 9 , 10. aunque varios estudios han examinado aspectos del desarrollo de tejido en el ovario pupa10,11,12,13, echara la diferenciación y organización espacial tipos de células ovario durante el desarrollo pupal. En particular, durante este período ocurre la especificación de Eve. Este protocolo describe un método para la disección y la coloración ovarios pupas en puntos del tiempo deseado, una técnica que puede utilizarse en experimentos del curso del tiempo que analizan el desarrollo pupal ovario en detalle de las larvas a la etapa adulta.
Para tener en cuenta la inaccesibilidad del ovario dentro del abdomen pupa pupa, tamaño pequeño y transparencia, este protocolo utiliza herramientas tales como una pipeta de Pasteur de punta fina a medida para remover el tejido abdominal grasa corporal obstaculizar el acceso del anticuerpo a la ovarios. Un cubreobjetos claro, cámara utilizado durante el anticuerpo coloración ofrece una mayor visibilidad de las pupas y una plataforma más para mecer los ovarios en una "Nutator." Basado en un protocolo para disecciones de ovario larvas de Maimon y Gilboa14, una concentración relativamente alta de Triton X-100 se ha empleado en los primeros pasos del procedimiento de tinción para maximizar el acceso permeabilización y el anticuerpo de membrana de la célula a la células ováricas.

<table><tbody><tr><td>Dumont #5 Forceps, biology</td><td>Fine Scientific Tools</td><td>11252-20</td><td></td></tr><tr><td>Nunc Lab-Tek Chambered Coverglass</td><td>Thermo Fisher Scientific</td><td>155383</td><td></td></tr><tr><td>Dissection microscope</td><td>Nikon</td><td>SMZ-10A</td><td></td></tr><tr><td>Confocal Microscope</td><td>Carl Zeiss</td><td>LSM 700</td><td></td></tr><tr><td>Analysis software</td><td>Carl Zeiss</td><td>Zen</td><td></td></tr><tr><td>9 Depression Glass Spot Plates</td><td>Pyrex</td><td>7220-85</td><td></td></tr><tr><td>Pasteur pipet</td><td>Fisher Scientific</td><td>13-678-6B</td><td></td></tr><tr><td>Pasteur pipet bulb</td><td>Various vendors</td><td></td><td></td></tr><tr><td>Bunsen burner</td><td>Various vendors</td><td></td><td></td></tr><tr><td>Fisherfinest Premium Frosted Microscope Slides</td><td>Thermo Fisher Scientific</td><td>12-544-2</td><td></td></tr><tr><td>22 x 22 mm glass coverslips No 1</td><td>VWR</td><td>48366-067</td><td></td></tr><tr><td>Dapi Fluoromount-G</td><td>SouthernBiotech</td><td>0100-20</td><td></td></tr><tr><td>Double-sided tape</td><td>Scotch</td><td></td><td></td></tr><tr><td>Nutator</td><td>Clay Adams</td><td></td><td></td></tr><tr><td>Fine brush #0, #3-#5</td><td>Various vendors</td><td></td><td></td></tr><tr><td>Gilson Pipetman Starter Kit</td><td>Thomas Scientific</td><td>F167300</td><td>Contains p20, p200, p1000 pipettors</td></tr><tr><td>16% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Dilute to 4% paraformaldehyde in 1x PBS</td></tr><tr><td>Triton</td><td>Sigma-Aldrich</td><td>9002-93-1</td><td></td></tr><tr><td>10x PBS</td><td>Ambion</td><td>AM9624</td><td>Dilute to 1x PBS</td></tr><tr><td>Normal Goat Serum</td><td>Jackson ImmunoResearch</td><td>5000121</td><td>Dilute to 10% normal goat serum in PBST with 0.5% Triton concentration</td></tr><tr><td>Primary antibodies (in protocol: 7G10 anti-Fasciclin III diluted 1:250, rabbit anti-phosphohistone H3 diluted 1:1000)</td><td>Various vendors (in protocol: Developmental Studies Hybridoma Bank, Millipore)</td><td></td><td>Dilute in PBST with 0.5% Triton concentration</td></tr><tr><td>Secondary antibodies (in protocol: Alexa-546, FITC-conjugated anti-rabbit serum)</td><td>Various vendors (in protocol: Molecular Probes, Jackson ImmunoResearch Laboratories, Inc.)</td><td></td><td>Dilute in PBST with 0.5% Triton concentration</td></tr><tr><td>Fly vials</td><td>Denville Scientific</td><td>V9406</td><td></td></tr><tr><td>Cotton Balls, For Wide Vials</td><td>Genesee Scientific</td><td>51-102W</td><td></td></tr><tr><td>Yeast, Bakers Dried Active</td><td>MP Biomedicals</td><td>101400</td><td></td></tr><tr><td>Fly food</td><td>Produced in laboratory</td><td></td><td>Mixture of water, brewer&#039;s yeast, cornmeal, molasses, agar, EtOH, penicillin, methyl 4-hydrobenzoate, and propionic acid</td></tr><tr><td>Male and female &lt;em&gt;Drosophila&lt;/em&gt; flies (genotype used in protocol: yw; P[Fz3-RFP, w+]/TM2)</td><td>Bloomington Drosophila Stock Center</td><td></td><td></td></tr></tbody></table>

dissection and staining of drosophila pupal ovaries

A diferencia de ovarios de Drosophila adultos, pupas ovarios son relativamente difíciles de acceder y examinar debido a su pequeño tamaño, transparencia y encerrar dentro de una envoltura pupal. El reto de disección de ovarios pupas también radica en su ubicación física dentro de la pupa: los ovarios están rodeados por las células del cuerpo de la grasa dentro del abdomen pupal, y estas células de grasa debe ser removidas para permitir para la tinción de anticuerpos adecuada. Para superar estos desafíos, este protocolo utiliza pipetas de Pasteur modificado para requisitos particulares para extraer las células de la grasa corporal del abdomen pupal. Por otra parte, un cubreobjetos cámara se utiliza en lugar de un tubo de microcentrífuga durante el proceso de tinción para mejorar la visibilidad de las pupas. Sin embargo, a pesar de estas y otras ventajas de las herramientas utilizadas en el presente Protocolo, ejecución acertada de estas técnicas todavía puede implicar varios días de práctica debido al pequeño tamaño de los ovarios de la pupas. Las técnicas descritas en este protocolo se podrían aplicar a los experimentos de curso de tiempo en el que los ovarios se analizan en diversas etapas de desarrollo pupal.

Ejecución exitosa de este procedimiento debe resultar anticuerpos clara coloración que revela la estructura y organización celular de un ovario pupal de Drosophila . Inmunohistoquímica que se describe en este protocolo puede utilizarse para identificar tipos celulares comúnmente teñidos en ovarios de larvarios y adultos. Las células del tallo pupa derivado de enjambre de células18 (descritas por Fasciclin III en blanco) se muestran en la <strong cl...

Watch this Scientific Journal Video about Dissection and Staining of Drosophila Pupal Ovaries at JoVE.com

Dissection and Staining of Drosophila Pupal Ovaries

El ovario del Drosophila es un sistema modelo excelente para estudiar el desarrollo del nicho de células madre. Aunque se han publicado métodos para disecar larvarios y adultos de los ovarios, disecciones de ovario pupa requieren técnicas diferentes que no han sido publicadas en detalle. Aquí describiremos un protocolo de disección, tinción y montaje pupas ovarios.

Disección y la coloración de los ovarios Pupal de Drosophila

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

dissection-and-staining-of-drosophila-pupal-ovaries

Research

JoVE Journal

Developmental Biology

Dissection and Staining of Drosophila Pupal Ovaries

9.3K vistas.  Columbia University. El ovario del Drosophila es un sistema modelo excelente para estudiar el desarrollo del nicho de células madre. Aunque se han publicado métodos para disecar larvarios y adultos de los ovarios, disecciones de ovario pupa requieren técnicas diferentes que no han sido publicadas en detalle. Aquí describiremos un protocolo de disección, tinción y montaje pupas ovarios.

Video: Dissection and Staining of Drosophila Pupal Ovaries

Dissection of Drosophila Ovaries

La disección de los ovarios Drosophila

Investigación

Biología

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.

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 ...

Biología del Desarrollo

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

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

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

Visualización de los efectos del daño oxidativo en las cámaras de huevos de Drosophila usando imágenes en vivo

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

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

Imágenes confocales y de superresolución del tráfico intracelular polarizado y la secreción de proteínas de la membrana basal durante la oogénesis de Drosophila

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

La disección y la tinción de Drosophila Larvas Ovarios

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Drosophila Pupal Abdomen Immunohistochemistry

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

Drosophila Pupal Abdomen Immunohistochemistry

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

Drosophila pupa inmunohistoquímica Abdomen

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

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

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

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

La disección y la inmunotinción de imaginal discos de<em&gt; Drosophila melanogaster</em

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

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Estudiar mitocondrial Estructura y función en<em&gt; Drosophila</em&gt; Los ovarios

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Disección y tinción de gotas de lípidos de los ovenocitos en larvas de Drosophila

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

Disección, fijación y visualización del Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...

Disección y la coloración de los ovarios Pupal de Drosophila

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