Agradecemos a Sean B. Carroll y Thomas Werner para proporcionar acciones mosca, Naoyuki fusible para el equipo, Byung Seok Jin para su asistencia en rodaje, Kiyokazu Agata para tutoría y Elizabeth Nakajima para la edición de inglés. Este trabajo fue apoyado por KAKENHI 17K 19427 y Takeda Science Foundation.

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M., Butts, J. C., Ordway, A., Vellky, J. E., Rebeiz, M., Williams, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+origination+and+diversification+of+a+dimorphic+gene+regulatory+network+through+parallel+innovations+in+cis+and+trans.">The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans.</a> PLoS Genet. 11 (4), e1005136 (2015).</li><li>Gompel, N., Prud'homme, B., Wittkopp, P. J., Kassner, V. A., Carroll, S. 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Cell. 34 (5), 520-531 (2015).</li><li>Koshikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancer+modularity+and+the+evolution+of+new+traits.">Enhancer modularity and the evolution of new traits.</a> Fly. 9 (4), 155-159 (2015).</li><li>Koshikawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+of+cis-regulatory+activities+underlies+novel+domains+of+wingless+gene+expression+in+Drosophila.">Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila.</a> Proc Natl Acad Sci U S A. 112 (24), 7524-7529 (2015).</li><li>McGregor, A. 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=Morphological+evolution+through+multiple+cis-regulatory+mutations+at+a+single+gene.">Morphological evolution through multiple cis-regulatory mutations at a single gene.</a> Nature. 448 (7153), 587-590 (2007).</li><li>Tanaka, K., Barmina, O., Kopp, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+mechanisms+underlie+the+evolutionary+diversification+of+Drosophila+sex+combs.">Distinct developmental mechanisms underlie the evolutionary diversification of Drosophila sex combs.</a> Proc Natl Acad Sci U S A. 106 (12), 4764-4769 (2009).</li><li>Werner, T., Koshikawa, S., Williams, T. M., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+novel+wing+colour+pattern+by+the+Wingless+morphogen.">Generation of a novel wing colour pattern by the Wingless morphogen.</a> Nature. 464 (7292), 1143-1148 (2010).</li><li>Wittkopp, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraspecific+polymorphism+to+interspecific+divergence:+genetics+of+pigmentation+in+Drosophila.">Intraspecific polymorphism to interspecific divergence: genetics of pigmentation in Drosophila.</a> Science. 326 (5952), 540-544 (2009).</li><li>Lawrence, P. A., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+in+the+wing+of+Drosophila:+a+study+of+the+engrailed+gene.">Compartments in the wing of Drosophila: a study of the engrailed gene.</a> Dev Biol. 50 (2), 321-337 (1976).</li><li>Bainbridge, S. P., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+the+metamorphosis+of+Drosophila+melanogaster.">Staging the metamorphosis of Drosophila melanogaster.</a> J. Embryol. Exp. Morphol. 66, 57-80 (1981).</li><li>Ashburner, M., Golic, K. G., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Laboratory Handbook. , (2005).</li><li>Fukutomi, Y., Matsumoto, K., Agata, K., Funayama, N., Koshikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pupal+development+and+pigmentation+process+of+a+polka-dotted+fruit+fly,+Drosophila+guttifera+(Insecta,+Diptera).">Pupal development and pigmentation process of a polka-dotted fruit fly, Drosophila guttifera (Insecta, Diptera).</a> Dev Genes Evol. 227 (3), 171-180 (2017).</li><li>Koshikawa, S., Fukutomi, Y., Matsumoto, K., Sekimura, T., Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+guttifera+as+a+model+system+for+unraveling+color+pattern+formation.">Drosophila guttifera as a model system for unraveling color pattern formation.</a> Diversity and evolution of butterfly wing patterns: an integrative approach. , (2017).</li><li>True, J. R., Edwards, K. A., Yamamoto, D., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+wing+melanin+patterns+form+by+vein-dependent+elaboration+of+enzymatic+prepatterns.">Drosophila wing melanin patterns form by vein-dependent elaboration of enzymatic prepatterns.</a> Curr Biol. 9 (23), 1382-1391 (1999).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Izumitani, H. F., Kusaka, Y., Koshikawa, S., Toda, M. J., Katoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeography+of+the+Subgenus+Drosophila+(Diptera:+Drosophilidae):+evolutionary+history+of+faunal+divergence+between+the+Old+and+the+New+Worlds.">Phylogeography of the Subgenus Drosophila (Diptera: Drosophilidae): evolutionary history of faunal divergence between the Old and the New Worlds.</a> PLoS ONE. 11 (7), e0160051 (2016).</li><li>Resh, V. H., Card&#233;, R. 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Los autores no tienen conflicto de interés.

Describimos aquí los protocolos para la definición de etapas de pupal, eliminación del pupario de observación detallada, duración de etapas de pupal de medición medición de la intensidad de manchas negras sobre un ala en D. guttifera. Estos protocolos pueden ser aplicados para muchos Drosophila y relacionados con mosca de especies, especialmente con la pigmentación de la mariposa.
Profunda observación y descripción de los eventos del desarrollo más detallados permi...

Algunos de los rasgos morfológicos de Drosophila se diversifican entre especies1,2,3,4,5. Podemos abordar la cuestión de la diversidad morfológica cómo surge mediante la comparación de los mecanismos de generación de estas morfologías. Ejemplos de tales morfologías son tricomas larvarias, peines sexuales para adultos, aparato genital externo, pigmentación abdominal y ala pigmentación6,7,8,9, 10 , 11 , 12 , 13 , 14 , 15. para estudiar las diferencias morfológicas entre los adultos, observación y análisis de las etapas de pupas son importantes, porque el destino de rasgos adultos se determina en las últimas etapas larvales y morfogénesis posterior procede durante el período de pupa.
En estudios de Biología del desarrollo de Drosophila melanogaster, 'horas APF' (horas después de la formación pupal) es el método común para indicar una etapa pupal16. Este sistema emplea tiempo absoluto después de la formación pupal y es muy conveniente para los experimentos de la rutina. Sin embargo, velocidad de desarrollo puede diferir entre pupas, puede verse afectada por pequeñas diferencias genéticas, epigenéticas o microambiental, y por lo tanto, con el mismo tiempo absoluto después de la formación pupal no garantiza que las pupas son al mismo etapa de desarrollo. En muchos casos, son preferibles para comparar a varios individuos etapas definidas por características morfológicas. Sobre todo, una comparación entre especies requiere precisa puesta en escena y la comparación entre correspondientes (homólogos).
Bainbridge y Bownes17 reconoce 20 etapas de pupal (P1 a P15(ii)) basado en las características morfológicas de pupas de Drosophila melanogaster . Esta puesta en escena es el sistema más ampliamente utilizado de la puesta en escena del desarrollo morfológico18. En un estudio anterior, se realizó estadificación pupal de Drosophila guttifera para establecer una base para ala pigmentación estudios19. D. guttifera tiene un patrón de lunares negros en sus alas y es una de las especies modelo de ala pigmentación formación20. Aunque nos referimos a los criterios morfológicos descritos en el Bainbridge y 'Bownes investigación17, directamente midiendo duraciones de etapa observaciones serie19, en lugar de utilizar Bainbridge y 'Bownes estimación de las duraciones de la fase de de frecuencia observada. Aquí describimos el método de puesta en escena pupal y medición de duración de etapas de pupal de Drosophila en Fukutomi et al19.
Para estudiar el mecanismo del desarrollo de la pigmentación de ala, que necesitamos saber cuando en estadios de pupas o adulto se produce la pigmentación. Fukutomi et al. 19 había cuantificado densidades ópticas (ODs) de la pigmentación durante etapas de pupas y adulto por análisis de imágenes de imágenes de ala. La pigmentación de las alas de Drosophila se piensa para ser causado por la acumulación de melanina negra21. Para la cuantificación de Sao, se utilizaron imágenes de escala de grises y ImageJ software (https://imagej.nih.gov/ij/)22 . Para reconocer y cuantificar la pigmentación específica de lugar (ΔOD), restamos el OD fuera de un lugar de la do dentro de un lugar. Para hacer este método reproducible y objetiva, se deben determinar los lugares de medición de OD con venas de las alas como puntos de referencia. En este artículo, describimos en detalle este método de cuantificación de la pigmentación de ala de Drosophila guttifera.

<table>
	<tbody>
		<tr>
			<td>Drosophila guttifera</td>
			<td>The Drosophila Species Stock Center at the U.C. San Diego</td>
			<td>15130-1971.10</td>
			<td>Drosophila guttifera, a fruit fly species used in this article</td>
		</tr>
		<tr>
			<td>Plastic vial</td>
			<td>Hightech</td>
			<td>MKC-30</td>
			<td>Plastic vial, for fly stock maintenance</td>
		</tr>
		<tr>
			<td>Buzz plugs vial and bottle closures for glass vials</td>
			<td>Fisher Scientific</td>
			<td>AS-271</td>
			<td>Cellulose plug, for fly stock maintenance</td>
		</tr>
		<tr>
			<td>White soft sugar</td>
			<td>Mitsui Sugar</td>
			<td>J-500g</td>
			<td>White soft sugar, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Corn flour</td>
			<td>Nippon Flour Mills</td>
			<td>F</td>
			<td>Corn flour, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Corn grits - C</td>
			<td>Nippon Flour Mills</td>
			<td>GC</td>
			<td>Corn grits - C, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Matsuki Kanten Sangyo</td>
			<td>No.602</td>
			<td>Agar powder, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Dry beer yeast</td>
			<td>Asahi Food &#38; Healthcare</td>
			<td>Y2A</td>
			<td>Dry beer yeast, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Butyl p-hydroxybenzoate</td>
			<td>Nacalai Tesque</td>
			<td>06327-02</td>
			<td>Butyl p-hydroxybenzoate, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Wako</td>
			<td>057-00456</td>
			<td>Ethanol, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Flat bottom microtube</td>
			<td>Ina Optica</td>
			<td>CF-0150</td>
			<td>1.5 mL microtube, for collecting pupae</td>
		</tr>
		<tr>
			<td>CAPSULEFUGE</td>
			<td>Tomy</td>
			<td>PMC-060</td>
			<td>Mini microcentrifuge, for collecting pupae</td>
		</tr>
		<tr>
			<td>Sterilized Schale NB</td>
			<td>Sansei Medical</td>
			<td>01-013</td>
			<td>Plastic Petri dish (diameter 90 mm x height 15 mm)</td>
		</tr>
		<tr>
			<td>Serum tube rack</td>
			<td>Iwaki</td>
			<td>9796-050</td>
			<td>Used as a moist chamber, for observation of pupa</td>
		</tr>
		<tr>
			<td>Corning Falcon Easy-Grip tissue culture dish</td>
			<td>Corning</td>
			<td>353001</td>
			<td>Plastic Petri dish (diameter 35 mm x height 10 mm)</td>
		</tr>
		<tr>
			<td>Falcon standard tissue culture dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td>Plastic Petri dish (diameter 60 mm x height 15 mm)</td>
		</tr>
		<tr>
			<td>Push-pin</td>
			<td>Kokuyo</td>
			<td>51233709</td>
			<td>Push-pin, for making pinholes on the microtube lid</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td>Stereomicroscope, for morphological observation</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Olympus</td>
			<td>DSE-330-A</td>
			<td>Digital camera, for imaging</td>
		</tr>
		<tr>
			<td>NICETACK double sided tape</td>
			<td>Nichiban</td>
			<td>NW-15SF</td>
			<td>Double sided tape, for removing puparium</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Forceps, for removing puparium</td>
		</tr>
		<tr>
			<td>Van Gogh VISUAL Paint brush</td>
			<td>Talens Japan</td>
			<td>GWVR-#5/0</td>
			<td>Paint brush, for removing puparium</td>
		</tr>
		<tr>
			<td>Greiner CELLSTAR 12 well cell culture plate</td>
			<td>Merck</td>
			<td>665-180</td>
			<td>12-well cell culture plate, for measuring durations of pupal periods</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td>NaCl, for PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Nacalai Tesque</td>
			<td>285-14</td>
			<td>KCl, for PBS</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;12H2O</td>
			<td>Wako</td>
			<td>196-02835</td>
			<td>Na2HPO4&#183;12H2O, for PBS</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Nacalai Tesque</td>
			<td>28721-55</td>
			<td>KH2PO4, for PBS</td>
		</tr>
		<tr>
			<td>Stepped Neutral Density (ND) Filter 0.04 - 3.0</td>
			<td>Edmund Optics</td>
			<td>64-384</td>
			<td>Stepped density filter, for calibration of pigmentation measurement</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td>1.8.0-101</td>
			<td>ImageJ software, for measurement of intensity of black spots on a wing (https://imagej.nih.gov)</td>
		</tr>
		<tr>
			<td>FINE FROST glass slide</td>
			<td>Matsunami Glass Ind</td>
			<td>FF-001</td>
			<td>Glass slide, for measurement of intensity of black spots on a wing</td>
		</tr>
		<tr>
			<td>Square microscope cover glass 18 x 18</td>
			<td>Matsunami Glass Ind</td>
			<td>C018181</td>
			<td>Cover slip, for measurement of intensity of black spots on a wing</td>
		</tr>
	</tbody>
</table>

methods for staging pupal periods and measurement of wing pigmentation of drosophila guttifera

Diversificada especies de Drosophila (mosca de la fruta) proporcionan oportunidades para el estudio de mecanismos de desarrollo y los cambios genéticos responsables de cambios evolutivos. En particular, la etapa adulta es una rica fuente de caracteres morfológicos para la comparación interespecífica, incluyendo ala pigmentación comparación. Para estudiar las diferencias del desarrollo entre especies, observación detallada y adecuada puesta en escena se requiere para la comparación exacta. Aquí describimos protocolos de puesta en escena de períodos pupas y cuantificación de la pigmentación de ala en una lunares mosca de la fruta Drosophila guttifera. En primer lugar, describimos el método de observación morfológica detallada y definición de etapas de pupal basadas en morfologías. Este método incluye una técnica para quitar del pupario, que es la carcaza externa quitinosa de la pupa, para permitir la observación detallada de la morfología pupal. En segundo lugar, describimos el método para medir la duración de etapas pupas definidas. Finalmente, describimos el método para la cuantificación de la pigmentación ala basada en análisis de imágenes mediante imágenes digitales y el software ImageJ. Con estos métodos, podemos establecer una base sólida para comparar procesos de desarrollo de rasgos adultos durante etapas de pupal.

El período pupal de D. guttifera se divide en 17 etapas (P1 - P15(ii); imágenes de representante de tres etapas (P1, P5 - 6, P10) se muestran la figura 3y 17 todas las etapas se ilustran en la figura 4). Aunque Bainbridge y Bownes17 reconocidas 20 etapas en D. melanogaster, algunas de estas etapas pueden no aplicarse a D. guttifera. El orden de los dos eventos del desarrollo, l...

Watch this Scientific Journal Video about Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera at JoVE.com

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Se describen los protocolos de presentar períodos de pupas y medición de la pigmentación de ala de Drosophila guttifera . Puesta en escena y cuantificación de pigmentación proporcionan una base sólida para el estudio de los mecanismos del desarrollo de rasgos adultos y permiten la comparación interespecífica de desarrollo rasgo.

Métodos para pupa períodos de prueba y medición de pigmentación de ala de Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

methods-for-staging-pupal-periods-measurement-wing-pigmentation

Research

JoVE Journal

Biology

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

11.5K vistas.  Kyoto University. Se describen los protocolos de presentar períodos de pupas y medición de la pigmentación de ala de Drosophila guttifera . Puesta en escena y cuantificación de pigmentación proporcionan una base sólida para el estudio de los mecanismos del desarrollo de rasgos adultos y permiten la comparación interespecífica de desarrollo rasgo.

Video: Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

De alta resolución de medición del comportamiento de olor-Driven en larvas de Drosophila

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Investigación

Biología

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Los métodos electrofisiológicos para la grabación de los potenciales sinápticos de la UNM de larvas de Drosophila

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

La disección en vivo de Drosophila Los embriones: métodos simplificados para la detección colecciones mutantes tinción con anticuerpos

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

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

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

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

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

Microdisección láser aplicada a perfiles de expresión génica de subconjunto de las células de la Drosophila Ala Disco

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

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 ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Medición de la esperanza de vida en<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Un método mejorado para la rápida y precisa medición de rendimiento en vuelo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imaging A través de la envoltura pupal de<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

La medición de la tasa metabólica en<em&gt; Drosophila</em&gt; Mediante respirometría

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

Ex vivo Cultura de Drosophila Pupal Testis y individuales masculinos quistes línea germinal: Disección, Imaging y Tratamiento farmacológico

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...