Questo protocollo è attualmente utilizzato nel nostro laboratorio ed è stato affinato negli anni da molte persone tra dottori David Sharp, Mijung Kwon, Patrizia Sommi, Dhanya Cheerambathur. Ringraziamo il dottor Bill Sullivan (UCSC), che ci ha fornito ottimi consigli sulla manipolazione e la microiniezione di primi embrioni di Drosophila quando il nostro lavoro in questo sistema era in fase di avvio (rif. 13). Ringraziamo tutti i membri del laboratorio Scholey. Il nostro lavoro sulla mitosi in Drosophila è sostenuto da NIH concedere GM55507.

Ricette: Succo d&#39;uva piastre: <ul><li> 5.5g bacto agar </li><li> 14,5 g destrosio o glucosio </li><li> 7,15 g di saccarosio </li><li> 45 ml di succo concentrato d&#39;uva (100% di succo). </li><li> 204,5 ml di H 2 0 </li><li> 625 microlitri 10N NaOH </li></ul> Mescolare tutti gli ingredienti e forno a microonde fino a ebollizione. Aggiungere 2,8 ml di acido mix (mix di acido: 20,9 ml di acido propionico, acido fosforico 2,1 ml, 27 ml di H 2 0) Mescolare e versare su 35 piatti millimetri Petri. Lasciate solidificare a temperatura ambiente per uno o due giorni. Se i pannelli non saranno utilizzati presto, sigillare con parafilm e conservare a 4 ° C (permette di equilibrare a RT prima di usare). Lievito di pasta: Sciogliere circa un cucchiaino di lievito (Sigma YSC2, lievito di Saccharomyces cerevisiae tipo II) in acqua per formare una pasta spessa. Mettete una piccola quantità di questo su ogni piatto succo d&#39;uva appena prima dell&#39;uso. G-PEM buffer: <ul><li> 80 mm Na-TUBI, pH 6,9 </li><li> 1 mM MgCl 2 </li><li> 1 mM EGTA </li><li> 1 mM GTP </li></ul> Iniezione buffer: <ul><li> 150 mM K-aspartato </li><li> 10 mM K-fosfato </li><li> 20 mM imidazolo, pH 7,2 </li></ul> Eptano colla: Srotolare il nastro doppio adesivo e mettere in un flacone da 100 ml, aggiungere circa 50 ml di eptano, sigillare la bottiglia, e il rock per diversi giorni. Durante l&#39;utilizzo, aggiungere eptano se la colla è troppo spessa. Camera di disidratazione: Prendete un piatto di 100 millimetri di Petri, mettere una parte di un piatto 35 millimetri all&#39;interno per fare una &quot;tabella&quot; e aggiungere Drierite (solfato di calcio anidro) intorno ad esso in modo che l&#39;altezza del Drierite non è superiore al &quot;tavolo&quot; e copertura. Il coprioggetto con embrioni saranno messi su questo &quot;tavolo&quot; per la disidratazione prima dell&#39;iniezione. E &#39;una buona idea di utilizzare almeno alcune Drierite indicazione e di cambiarlo quando ha cambiato colore. Protocollo: Questo protocollo può essere utilizzato per l&#39;iniezione di qualsiasi reagente solubile in embrione di Drosophila sinciziale: per esempio, o una proteina fluorescente per l&#39;osservazione o di un inibitore della proteina bersaglio o entrambi. Ottenere tutto pronto: 1 .- 2 o 3 giorni prima dell&#39;esperimento, fanno piatti succo d&#39;uva e impostare laici gabbia: Prendere una bottiglia di plastica (6 oz, fondo rotondo da Applied Scientific Drosophila), tagliare due piccoli fori (circa 1 cm 2) sui lati opposti del bottiglia e riempire con un pezzo di cotone (questo permette all&#39;aria di circolare), toccare le mosche nella bottiglia e coprire con un piatto di succo d&#39;uva. Mantenere a 25 ° C (o temperatura ambiente), mosche dovrebbero definire gli embrioni per circa 10 giorni. 2 .- Almeno un giorno prima l&#39;esperimento tirare aghi per l&#39;iniezione, si usa un ago Kopf / Pipettare modello estrattore 720 e filamenti sottili pareti di vetro (World Strumenti di precisione tw100f). Per l&#39;iniezione di tubulina fluorescente, mantenere gli aghi a 4 ° C per evitare che la temperatura indotta polimerizzazione nella ago. Tubulina preparazione: Nota: Tutti i lavori con la tubulina dovrebbe essere fatto a 4 ° C per prevenire la polimerizzazione. <ol><li> Prendere tubulina etichettati dal freezer e metterlo in ghiaccio per 5 a 15 minuti. </li><li> Diluire con G-PEM tampone (4 a 10 volte a seconda della intensità richiesta). </li><li> Centrifugare a 13k rpm a 4 ° C per 10-15 minuti. </li><li> Carico 1-2 microlitri in ago. </li></ol> Anticorpi o inibitori chimici: Preparare gli aghi come per tubulina, utilizzando una concentrazione appropriata (questo può avere bisogno di essere testati). L&#39;anticorpo può essere nel buffer di iniezione, G-PEM buffer o PBS. Se un altro tampone è necessario, questo buffer dovrà essere iniettato come controllo per assicurarsi che questo buffer non causa alcun danno per conto suo. Nota: tubulina inibitore e fluorescenti possono essere miscelati e iniettati insieme, se la concentrazione lo consentono. Altrimenti, iniettare il tubulina fluorescenti e attendere 5 a 10 minuti prima di iniettare l&#39;inibitore. Quando si esegue una doppia iniezione, iniziare con più embrioni, dal momento che un minor numero di embrioni si riprenderà normalmente. Raccolta degli embrioni: <ol><li> Mettere un nuovo piatto succo d&#39;uva sulla gabbia laici. </li><li> Rimuovi questo piatto dopo un&#39;ora, questa è la prima collezione della giornata e spesso non è molto buona. Può essere scartato. </li><li> Cambia la piastra ogni ora per tenere raccolta embrioni per un&#39;ora ciascuno. </li></ol> Coprioggetto preparazione: <ol><li> Mettere un coprioggetti 50 x 22 millimetri su un lato di un vetrino da microscopio. Nastro i quattro angoli alla diapositiva in modo che non si muove. </li><li> Utilizzando un cotone con punta applicatore, mettere uno strato di colla eptano in una riga sul coprioggetto (la colla non deve essere viscoso e deve asciugare in pochi secondi, altrimenti aggiungere più eptano). </li><li> Mettete un pezzo didoppio nastro adesivo sul vetrino verso il lato del coprioggetto. </li></ol> Embrione preparazione: Nota: Gli embrioni devono essere esposte circa 2 ore dopo l&#39;inizio della raccolta, in modo da iniziare la seguente procedura che consente un tempo sufficiente per finirli entro questo arco di tempo. <ol><li> Con un pennello inumidito con attenzione raccogliere gli embrioni dalla piastra di succo d&#39;uva e il luogo in doppio nastro adesivo sul vetrino. </li><li> Utilizzando la parte esterna delle pinzette, rotolare gli embrioni sopra il nastro doppio adesivo fino a quando il corion (la membrana esterna) si rompe. </li><li> Prendete l&#39;embrione con delicatezza ribaltamento del corion così si attacca alla pinzetta e posizionarlo sulla colla eptano sul vetrino con il lato lungo del parallelo embrione al lato lungo del vetrino coprioggetto. Impostare 10-20 embrioni in una riga. </li><li> Rimuovere il coprioggetto e posizionarlo nella camera di disidratazione per 3 a 8 minuti (questo dipende l&#39;umidità della stanza e la quantità da iniettare). </li><li> Mettere su una camera di metallo con grasso per vuoto. </li><li> Coprire gli embrioni con 700 Halocarbon olio per evitare ulteriori disidratazione. Gli embrioni sono ora pronti per l&#39;iniezione. </li></ol> Embrione iniezione: <ol><li> Trova gli embrioni sotto un obiettivo 16x. </li><li> Spostare gli embrioni di distanza, e trovare l&#39;ago senza spostare il piano focale. </li><li> Centro l&#39;ago e spostarlo senza spostarlo nella direzione xo y.. </li><li> Se l&#39;ago non è aperto, messo ai margini del vetrino nel campo di vista, ma non dove l&#39;ago sarà, e abbassare l&#39;ago stesso piano focale. Con molta attenzione spostare il coprioggetto fino a raggiungere l&#39;ago e delicatamente spezza aperto. Se l&#39;ago è aperto, passare al punto 5. (Aghi possono essere aperti con acido fluoridrico prima del riempimento). </li><li> Mettere gli embrioni in vista (ma non dove l&#39;ago sarà), abbassare l&#39;ago nel petrolio e assicurarsi di ottenere gocce di liquido bella dall&#39;ago. </li><li> Con attenzione ma costantemente spostare l&#39;embrione in l&#39;ago e iniettare una goccia in embrione e spostare l&#39;embrione distanza. (Oppure seguire le istruzioni del tuo apparato di iniezione). </li><li> Dopo che tutti gli embrioni sono stati iniettati, sono pronti per l&#39;osservazione di un microscopio confocale. </li></ol> Imaging: Immagine gli embrioni in un microscopio confocale con un obiettivo 60 o 100X: Note: <ol><li> L&#39;intervallo di tempo su una serie lasso di tempo dovrebbe essere al massimo di pochi secondi, dal momento che la mitosi nell&#39;embrione è molto veloce, a seconda del processo esatto da studiare. </li><li> Una serie di immagini 3D o di un singolo piano può essere acquisito a seconda del processo di interesse. </li><li> Per gli inibitori ad azione rapida, il trasferimento da microscopio iniezione per l&#39;imaging microscopio deve essere veloce. </li></ol><img src="/files/ftp_upload/1382/1382fig1.jpg" alt="figura 1" /> Figura 1: Studio della mitosi negli embrioni di Drosophila sinciziale. Nuclei nell&#39;embrione precoce subire divisioni rapido senza citodieresi, durante i cicli di 10 e 13 formano un monostrato a nuclei della corteccia. A. Schematica di embrione precoce con i nuclei di corteccia. Embrione può esprimere la GFP e / o RFP proteine ​​marcate e può essere microiniettati con altre proteine ​​marcate e / o inibitori. B. lapse imaging Tempo di embrioni che esprimono GFP-tubulina e RFP-istoni. Plot C. polo-polo di separazione per cicli di 11 , 12 e 13 in un embrione di tipo selvatico. Lunghezza del mandrino presenta periodi di lunghezza isometrica e periodi di allungamento, che sono molto coerenti e riproducibili. D. Dinamica dei microtubuli del fuso. L&#39;iniezione di bassa concentrazione di tubulina rodamina porta a macchie formato da una subunità di tubulina pochi, che può essere utilizzato per studiare la dinamica dei microtubuli.

<ol>
	<li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+dynamics+in+Drosophila.">Mitotic spindle dynamics in Drosophila.</a> Int Rev Cytol. 259, 139-172 (2007).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34, 1-15 (2002).</li><li>Morris, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+methods+for+analyzing+the+functions+of+kinesins+in+early+embryos.">Microinjection methods for analyzing the functions of kinesins in early embryos.</a> Methods Mol Biol. 164, 163-172 (2001).</li><li>Brust-Mascher, I., Sommi, P., Cheerambathur, D. K., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin-5-dependent+poleward+flux+and+spindle+length+control+in+Drosophila+embryo+mitosis.">Kinesin-5-dependent poleward flux and spindle length control in Drosophila embryo mitosis.</a> Mol Biol Cell. 20, 1749-1762 (2009).</li><li>Brust-Mascher, I., Civelekoglu-Scholey, G., Kwon, M., Mogilner, A., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+for+anaphase+B:+role+of+three+mitotic+motors+in+a+switch+from+poleward+flux+to+spindle+elongation.">Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation.</a> Proc Natl Acad Sci U S A. 101, 15938-15943 (2004).</li><li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+flux+and+sliding+in+mitotic+spindles+of+Drosophila+embryos.">Microtubule flux and sliding in mitotic spindles of Drosophila embryos.</a> Mol Biol Cell. 13, 3967-3975 (2002).</li><li>Cheerambathur, D. K., Brust-Mascher, I., Civelekoglu-Scholey, G., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+partitioning+of+mitotic+kinesin-5+crosslinkers+between+microtubule-bound+and+freely+diffusing+states.">Dynamic partitioning of mitotic kinesin-5 crosslinkers between microtubule-bound and freely diffusing states.</a> J Cell Biol. 182, 421-428 (2008).</li><li>Cheerambathur, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+an+anaphase+B+switch:+predicted+role+for+a+microtubule+catastrophe+gradient.">Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient.</a> J Cell Biol. 177, 995-1004 (2007).</li><li>Kwon, 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+chromokinesin,+KLP3A,+dives+mitotic+spindle+pole+separation+during+prometaphase+and+anaphase+and+facilitates+chromatid+motility.">The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility.</a> Mol Biol Cell. 15, 219-233 (2004).</li><li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+coordination+of+three+mitotic+motors+in+Drosophila+embryos.">Functional coordination of three mitotic motors in Drosophila embryos.</a> Mol Biol Cell. 11, 241-253 (2000).</li><li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bipolar+kinesin,+KLP61F,+cross-links+microtubules+within+interpolar+microtubule+bundles+of+Drosophila+embryonic+mitotic+spindles.">The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.</a> J Cell Biol. 144, 125-138 (1999).</li><li>Sharp, D. J., Rogers, G. C., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+dynein+is+required+for+poleward+chromosome+movement+during+mitosis+in+Drosophila+embryos.">Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos.</a> Nat Cell Biol. 2, 922-930 (2000).</li><li>Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+microtubule-sliding+motors+position+mitotic+centrosomes+in+Drosophila+early+embryos.">Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos.</a> Nat Cell Biol. 1, 51-54 (1999).</li><li>Waterman-Storer, C. M., Desai, A., Bulinski, J. C., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy,+a+method+to+visualize+the+dynamics+of+protein+assemblies+in+living+cells.">Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells.</a> Curr Biol. 8, 1227-1230 (1998).</li></ol>

Questo protocollo è relativamente semplice, tuttavia, ogni passo richiede pratica per assicurarsi che gli embrioni non siano danneggiati. Esperimenti di controllo attento sempre bisogno di essere fatto per garantire che i risultati affidabili si stanno ottenendo. Un buon modo per cominciare questo è microinjecting tubulina buffer o rodamina in embrioni di controllo che esprime GFP-tubulina per assicurarsi che la mitosi procede normalmente attraverso tutti i cicli in superficie dell&#39;embrione (cicli da 10 a 13). Neg...

microinjection techniques for studying mitosis in the drosophila melanogaster syncytial embryo

Questo protocollo descrive l&#39;uso di embrioni di Drosophila melanogaster sinciziale per lo studio mitosi 1. Drosophila è genetica utile con un genoma sequenziato, e può essere facilmente gestito e manipolato. Mutanti mitotici esistono molte, e vola transgenici che esprimono funzionale fluorescente (es. GFP) - proteine ​​mitotico tag sono stati e vengono generati. Espressione genica mirata è possibile con il sistema GAL4/UAS 2. L&#39;embrione di Drosophila primi svolge mitosi multipli molto rapidamente (durata del ciclo cellulare, ≈ 10 min). E &#39;adatto per l&#39;imaging mitosi, perché durante i cicli di 10-13, i nuclei si dividono rapidamente e in modo sincrono senza intervenire citocinesi sulla superficie dell&#39;embrione in un monostrato singolo appena sotto la corteccia. Questi nuclei si dividono rapidamente probabilmente usare gli stessi macchinari mitotico come le altre cellule, ma sono ottimizzate per la velocità, il checkpoint è generalmente ritenuto di non essere rigorosi, permettendo lo studio delle proteine ​​mitotico la cui assenza avrebbe causato l&#39;arresto del ciclo cellulare in cellule con un posto di blocco forte . Gli embrioni che esprimono proteine ​​GFP etichettati o microiniettati con le proteine ​​fluorescente può essere facilmente ripreso a seguire le dinamiche vivere (Fig. 1). Inoltre, gli embrioni possono essere microiniettati con funzione di blocco anticorpi o inibitori di proteine ​​specifiche per studiare l&#39;effetto della perdita o di perturbazione della loro funzione 3. Questi reagenti possono diffondere in tutto l&#39;embrione, raggiungendo molti fusi per produrre un gradiente di concentrazione di inibitore, che a sua volta in un gradiente di difetti paragonabile ad una serie alleliche di mutanti. Idealmente, se la proteina bersaglio è fluorescente, il gradiente di inibizione può essere direttamente visualizzato 4. Si presume che il più forte fenotipo è paragonabile al fenotipo nullo, anche se è difficile escludere formalmente la possibilità che gli anticorpi possono avere effetti dominante in rari casi, i controlli in modo rigoroso e cauta interpretazione deve essere applicata. Più lontano dal sito di iniezione, la funzione delle proteine ​​è solo parzialmente perso permettendo altre funzioni della proteina bersaglio a diventare evidente.

Watch this Scientific Journal Video about Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo at JoVE.com

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Questo protocollo descrive l&#39;uso di microiniezione e di imaging ad alta risoluzione nel<em&gt; Drosophila melanogaster</em&gt; Embrione sinciziale per studiare la mitosi.

Tecniche di microiniezione per Studiare Mitosi in Drosophila melanogaster Embryo sinciziale

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

microinjection-techniques-for-studying-mitosis-drosophila

Research

JoVE Journal

Biology

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

14.0K Views.  University of California, Davis. Questo protocollo descrive l'uso di microiniezione e di imaging ad alta risoluzione nel Drosophila melanogaster Embrione sinciziale per studiare la mitosi.

Video: Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissezione del sistema nervoso centrale larvali in Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Ricerca

Biologia

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Monitoraggio ad alta risoluzione video di locomozione per adulti in Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Registrazione elettrofisiologiche in Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Il monitoraggio delle funzioni cardiaca in larvali Drosophila melanogaster Per gli Studi fisiologici

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

Studiare Checkpoint mitotica illustrando un comportamento dinamico delle proteine ​​cinetocoro e Motion cromosoma in Living<em&gt; Drosophila</em&gt; Embrioni sinciziale

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

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.

Misura della durata della vita in<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 ...

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 attraverso il caso pupa di<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 ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

Maggiore Northern Blot rilevazione di piccoli RNA specie in<em&gt; Drosophila melanogaster</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.