Ringraziamo Nicki Fox e Aloisia Schmid per il loro contributo allo sviluppo di questi protocolli. Questo lavoro è stato supportato da un RO1 NIH accordare ai KZ, NS28182.

<ol>
	<li>Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+and+other+cell+types+in+whole-mount+Drosophila+embryos+and+larvae+using+antibody+probes.">Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes.</a> Methods Cell Biol. 44, 445-487 (1994).</li><li>Desai, C. J., Gindhart, J. G., Goldstein, L. S., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+tyrosine+phosphatases+are+required+for+motor+axon+guidance+in+the+Drosophila+embryo.">Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo.</a> Cell. 84, 599-609 (1996).</li><li>Sun, Q., Schindelholz, B., Knirr, M., Schmid, A., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+genetic+interactions+among+four+receptor+tyrosine+phosphatases+regulate+axon+guidance+in+Drosophila.">Complex genetic interactions among four receptor tyrosine phosphatases regulate axon guidance in Drosophila.</a> Molecular and cellular neurosciences. 17, 274-291 (2001).</li><li>Schmid, A., Schindelholz, B., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+RNAi:+a+method+for+evaluating+the+functions+of+gene+families+in+Drosophila.">Combinatorial RNAi: a method for evaluating the functions of gene families in Drosophila.</a> Trends in neurosciences. 25, 71-74 (2002).</li><li>Fox, A. N., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heparan+sulfate+proteoglycan+syndecan+is+an+in+vivo+ligand+for+the+Drosophila+LAR+receptor+tyrosine+phosphatase.">The heparan sulfate proteoglycan syndecan is an in vivo ligand for the Drosophila LAR receptor tyrosine phosphatase.</a> Curr Biol. 15, 1701-1711 (2005).</li></ol>

Ci auguriamo che questa dimostrazione video ha mostrato i nostri metodi per la dissezione dell&#39;embrione dal vivo e colorazione in modo sufficientemente dettagliato che adesso possono essere facilmente eseguito da una persona che ha una certa esperienza in Drosophila genetica e dell&#39;embriologia. Naturalmente, la pratica considerevole sarà ancora necessaria, soprattutto per gli stessi dissezioni. Nella nostra esperienza, ogni persona che impara i metodi di dissezione dal vivo crea un protocollo di dissez...

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<td>Borosilicate Tubing With Filament </td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>BF120-60-10</td>
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<td>Narishige model PC-10 needle puller</td>
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<td>Narishige</td>
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<td>Tubes pulled using a heater level of 58.9</td>
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live dissection of drosophila embryos: streamlined methods for screening mutant collections by antibody staining

Embrioni di Drosophila tra 14 e 17 stadi dello sviluppo embrionale può essere facilmente sezionato per generare &quot;filetto&quot; preparati. In questi preparativi, il sistema nervoso centrale funziona a metà, ed è affiancato dalle pareti del corpo. Molti fenotipi differenti sono stati esaminati utilizzando tali preparati. Nella maggior parte dei casi, i filetti sono stati generati da dissezione di anticorpi colorati fisso intero montaggio embrioni. Questi &quot;dissezioni fisso&quot; sono alcuni svantaggi, però. Si tratta di tempo per eseguire, ed è difficile per ordinare mutante (GFP-negativo) gli embrioni provenienti da stock nel quale sono mantenute le mutazioni più cromosomi bilanciatori GFP. Dal 2002, il nostro gruppo sta conducendo carenza e schermi espressione ectopica di identificare ligandi per i recettori orfani. Per fare questo, abbiamo sviluppato protocolli ottimizzato per la dissezione dell&#39;embrione dal vivo e colorazione degli anticorpi di raccolte contenenti centinaia di linee bilanciate. Abbiamo concluso che è molto più efficiente per esaminare fenotipi in grandi collezioni di scorte da dissezione dal vivo che da dissezione fisso. Utilizzando il protocollo qui descritto, un singolo individuo addestrato può schermo fino a 10 righe al giorno per fenotipi, esaminando 4-7 embrioni mutanti da ogni riga sotto un microscopio composto. Ciò permette l&#39;identificazione di mutazioni conferimento sottile, a bassa penetranza fenotipi, dato che fino a 70 hemisegments per linea sono segnati a forte ingrandimento 40X con un acqua-immersion lente.

Watch this Scientific Journal Video about Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining at JoVE.com

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

Noi descriviamo un protocollo semplificato per la generazione di &quot;filetto&quot; preparati di embrioni di Drosophila genotipi specifici. Questo protocollo permette l&#39;esecuzione efficiente di una varietà di schermi genetica. Inoltre permette una visualizzazione eccellente delle strutture nell&#39;embrione tardi.

Dissezione di vivere Drosophila Embrioni: Metodi semplificati per lo screening Collezioni Mutant dalla colorazione anticorpale

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

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JoVE Journal

Biology

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

14.2K Views.  California Institute of Technology. Noi descriviamo un protocollo semplificato per la generazione di "filetto" preparati di embrioni di Drosophila genotipi specifici. Questo protocollo permette l'esecuzione efficiente di una varietà di schermi genetica. Inoltre permette una visualizzazione eccellente delle strutture nell'embrione tardi.

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

Dissection of Drosophila Ovaries

Dissezione di ovaie Drosophila

Ricerca

Biologia

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissezione di 6,5 embrioni di topo dpc

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Metodo per la colorazione intero anticorpi Monte a Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila larvali NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

La preparazione di Drosophila Gli embrioni per Live-Imaging Utilizzando il protocollo goccia

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Preparazione dei cromosomi di Drosophila Zucche politene per l&#39;etichettatura di anticorpi

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In vivo centrifugazione di embrioni di Drosophila

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imaging verticale di Drosophila Embrioni

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

L&#39;interferenza di RNAi per iniezione in dsRNA Drosophila Embrioni

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

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.

Dissezione e colorazione di Drosophila Larvali ovaie

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