Les auteurs tiennent à remercier le Fonds de recherche du Président de l&#39;Université de Saint Louis attribué à WSN

Une. Entretien de Cages Population <ol><li> Maintenir les cages de la population à 25 ° C sur un cycle lumière-obscurité de 12 h. Tant que les groupes témoins et expérimentaux sont exposés aux mêmes conditions d&#39;éclairage, alors cette technique peut être réalisée dans un environnement de laboratoire standard. </li><li> Permettre aux femmes de nuit pondent des œufs sur les pommiers plaques jus-agar. </li><li> Recueillir les larves nouvellement écloses par le maintien des plaques avec des oeufs nouvellement déposés à 25 ° C pendant 24 heures. Placer une petite cuillerée de levure dans le centre de la plaque d&#39;attirer larves écloses. </li><li> Recueillir 1 er stade larvaire qui ont migré dans la pâte de levure dans le centre de la plaque de jus de pomme. Utilisez une spatule en métal pour la transférer sur une plaque de jus de pomme frais. La pâte de levure supplémentaires peuvent être ajoutées pour assurer une alimentation suffisante il est jusqu&#39;à ce que les tests sont réalisés. des plaques à base de jus de raisin peut également être utilisé à la place de plaques à base de jus de pomme. </li><li> Obtenir la fin de 2 ème-début 3e stade larvae en permettant 1er larvaires à l&#39;âge de 40-48 heures. Dans cette gamme les taux d&#39;alimentation sont constants 13. L&#39;âge peut être confirmé par un examen de la bouche des crochets comme il ya des changements distincts dans cette structure avec chaque mue larvaire. </li><li> Late 2 3 ème stade larvaire précoce-ème sont recueillis pour des analyses de comportement par doucement et longuement lavage des plaques à base de jus de pomme à l&#39;eau et à recueillir les larves sur un filtre à mailles. Les larves sont ensuite transféré dans une plaque de gélose. </li><li> Toutes les analyses sont effectuées en parallèle en utilisant des animaux témoins et expérimentaux. </li></ol> 2. Paradigm comportementale - Locomotion <ol><li> Placez un seul 3 ème stade larvaire sur un substrat de gélose à 2% dans une boîte de culture de tissu de 100 mm et permettre à la larve de s&#39;acclimater pendant 30 sec. Les larves utilisent leurs sclérites cephalopharyngeal (bouche crochets) pour propulser leur corps à travers la surface du substrat agar. Cela se fait sur une plaque séparée, parce que jet est plus difficile de visualiser les contractions du corps dans la solution de levure en tant que l&#39;animal est pratiquement la même couleur que la levure. </li><li> Observer et noter chaque postérieur au mouvement antérieur sur le substrat pour une période de 1 min. n = 20 pour chaque génotype. Pas plus de 10 animaux doivent être testés par plaque. </li></ol> 3. Comportement Paradigm - Alimentation <ol><li> Utilisation de mousses Inox # 5 pinces, transférer soigneusement la 3 ème stade larvaire de la plaque de gélose locomotrice au centre d&#39;une plaque d&#39;agar-remplie recouvert avec 5 ml de la solution de levure de boulanger 2% activé. Assurez-vous que la solution est homogène que la levure se dépose au fil du temps. Lorsque dans la solution de levure, les larves en grande partie rester en place et l&#39;alimentation, ce qui facilite l&#39;observation du comportement. Le taux de bouche contractions de crochet est en corrélation directe avec la quantité de nourriture ingérée 14. </li><li> Laisser larve de s&#39;acclimater pendant 30 sec. </li><li> Observer et noter le nombre debouche contractions de crochet pour une période de 1 min. n = 20 pour chaque génotype. </li></ol> 4. Les dissections Gut larvaires <ol><li> Faire une solution de formaldéhyde de fixation 4% EM-grade 1x solution saline tamponnée au phosphate (PBS) et placer dans un verre de tache 3 puits. </li><li> Disséquer soigneusement errant fin 3 e stade larvaire tripes dans un plat en verre de 3 puits en utilisant une solution PBS 1x, en s&#39;assurant que chaque fois que le proventricule est laissée intacte. Avec une pince, tenir l&#39;extrémité postérieure, et l&#39;autre, maintenir les crochets buccaux. Tout en maintenant le immobile extrémité postérieure, tirez doucement sur la bouche des crochets pour accéder aux tripes. Retirez tous les tissus associés (glandes salivaires, le cerveau, le corps gras, etc), puis transférer chaque intestin à l&#39;antenne 3 puits contenant le correctif de formaldéhyde. Errant 3 e stade larvaire sont utilisés parce que, à ce stade de développement, la larve cesse de s&#39;alimenter en préparation pour la nymphose, et le proventricule est effacé de la levure. </li> &lt;li&gt; Incuber tripes à 4 ° C pendant une nuit dans une boîte de culture de tissu opaque. </li><li> Avant de retirer la solution de formaldéhyde de fixation des puits, enlever le caecum gastrique et couper l&#39;intestin moyen ~ 150 um 2 du proventricule de sorte que les projections peuvent clairement être considérées sans obstacle. </li><li> Retirer formaldéhyde solution dans les puits et les remplacer par 1x PBT (1x PBS, 0,1% de sérum albumine bovine libre de protéase, X-100 0,1% de Triton) solution tampon. Laver soigneusement les tripes 6x pendant 10 min dans 1x PBT. Placer les échantillons de tissus sur un rotateur mécanique tout en faisant des lavages. </li><li> Incuber à 4 ° C pendant 1 h à 10 -6 M 5-HT pour améliorer la signalisation de la sérotonine. Laver soigneusement les tripes 6x pendant 10 min dans 1x PBT. Des études antérieures ont démontré que cette concentration de 5-HT exogène n&#39;affecte pas l&#39;architecture neuronale ou la densité de varicosités dans les analyses immunohistochimiques et augmente simplement le rapport signal à bruit de 15 à 16. </li><li> Incuber à 4 et# 176; C pendant une nuit dans l&#39;anticorps primaire anti-sérotonine (monoclonal dirigé à la souris ou au lapin polyclonal). Laver soigneusement les tripes 6x pendant 10 min dans 1x PBT. Placer les échantillons de tissus sur un rotateur mécanique tout en faisant des lavages. </li><li> Incuber à 4 ° C pendant 90 min dans l&#39;anticorps secondaire (Alexa Fluor 568 de chèvre anti-souris ou anti-IgG de lapin, dilution 1:400). Laver soigneusement les tripes 6x pendant 10 min dans 1x PBT. Placer les échantillons de tissus sur un rotateur mécanique tout en faisant des lavages. </li><li> Incuber dans du carbonate de sodium 4 mM pendant 10 min sur un agitateur mécanique, puis monter à 4% de carbonate de sodium gallate/20 mM de n-propyle, et vue en fluorescence. Le carbonate de sodium est utilisé pour convertir les échantillons à la mémoire tampon et le pH utilisé dans le milieu de montage médias. </li><li> Capturer des images d&#39;échantillons de tissus immunocolorées à un grossissement de 400X pour analyse. </li></ol> 5. Analyse de Neural Circuits <ol><li> fibres de neurites ont été quantifiés (nombre et taille des variceset le degré de ramification) en utilisant Neuroleucida et Neuroexplorer. Toutefois, cela peut également être réalisée manuellement ou en utilisant Simple Neurites Tracer (une application qui peut être téléchargée en ligne gratuit). </li><li> Tracer les projections de fibres individuelles du cerveau vers le proventricule et de quantifier le nombre de varicosités, les branches et le nombre de grandes varicosités par unité de longueur. Les fibres axonales saillie du cerveau sont regroupés dans le nerf recurrens et ne sont pas accessibles pour l&#39;analyse jusqu&#39;à ce qu&#39;ils atteignent le proventricule où ils se séparent et fasciculées. </li></ol>

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Développement aberrant du circuit stomatogastrique sérotoninergique, qui se produit à la fin de l&#39;embryogenèse, aura une incidence sur sa fonction mature. Les changements dans l&#39;architecture des neurites innervant l&#39;intestin peut être corrélée à la sortie fonctionnelle du circuit, qui est le taux d&#39;alimentation (mesurée par la bouche contractions de crochet dans une solution de levure) (Figure 1). L&#39;utilisation du système bipartite UAS-Gal4 chez la drosophile perme...

Drosophile est un système modèle extrêmement puissant pour étudier le développement des circuits neuronaux en raison du temps de génération rapide, à faible coût expérimental, et la capacité de manipuler et contrôler les facteurs génétiques et environnementaux. Neurogenèse, recherche de chemin neuronal et la synaptogenèse sont conservés entre l&#39;homme et la drosophile, par conséquent les mécanismes de création, le maintien et la modification des circuits de neurones sont conservés ainsi. Neurotransmetteurs classiques, tels que la sérotonine (5-hydroxytryptamine ou 5-HT) peuvent servir de facteurs de croissance avant l&#39;adoption de leurs rôles en tant que molécules de signalisation dans le circuit neuronal maturité 1-3 Des études antérieures ont montré que les niveaux perturbée 5-HT au cours de l&#39;embryogenèse modifier la connectivité des neurones matures 4. D&#39;autres ont montré que l&#39;application ectopique de la 5-HT aux neurones en culture Helisoma réprimer la croissance des neurites ainsi que la synaptogenèse 5-7. En Drosophila, développement des niveaux de 5-HT sont en relation inverse avec le nombre et la taille des varicosités ainsi que le degré de aborization, le long de la longueur des neurites en saillie à partir de l&#39;intestin antérieur du SNC 8. Neurotransmission sérotoninergique a été montré pour moduler les comportements alimentaires dans diverses espèces, y compris chez la drosophile 9.8. Le circuit d&#39;alimentation de la drosophile est un circuit relativement simple qui peut être utilisé comme modèle pour mettre en corrélation la sortie fonctionnelle (d&#39;alimentation) avec des modifications dans la mise au point des projections axonales à partir du cerveau de l&#39;intestin antérieur. Schoofs et al. ont montré que l&#39;alimentation des larves de drosophile est régie par des générateurs de motifs centraux qui influencent la musculature 10. Bien que l&#39;anatomie musculaire spécifique n&#39;est pas complètement élucidé, il a été démontré que le nerf antennaire, nerf maxillaire, et le nerf accessoire prothoracic sont responsables des cibles musculaires impliqués dans lecomportement alimentaire. La plupart des données concernant la musculature et le nerf anatomie de l&#39;alimentation des invertébrés est limité pour les larves de Calliphora. Le taux d&#39;alimentation des larves de deuxième stade peut être évaluée par la rétraction des sclérites cephalopharyngeal (bouche crochets), et est reproductible et à haut débit. Les plaques cephalopharyngeal sont innervés par les fibres du centre de neurones 5-HT par l&#39;intermédiaire du nerf frontal. Le proventricule, ou l&#39;intestin antérieur, est innervée par des fibres sérotoninergiques (recurrens nerf) qui fasciculés dans l&#39;intestin moyen et sont responsables de contractions de l&#39;intestin antérieur (figure 1) 11-12. Les changements dans la ramification axonale, et le nombre et la taille des varicosités le long de la longueur des neurites, peuvent être quantifiés en utilisant des techniques d&#39;immunohistochimie. La manipulation de la 5-HT neuronale au cours du développement, soit directement, soit indirectement, peut modifier la sortie fonctionnelle de ce circuit d&#39;alimentation, qui peut être évalué et en corrélation avec l&#39;évolution de la morphogy de l&#39;architecture des neurites.

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		<tr height="40">
			<td height="40" style="height:40px;width:217px;">Eclipse E-800 Microscope</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;"></td>
			<td style="width:251px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:217px;">Neuroleucida</td>
			<td style="width:175px;">MBF Biosciences</td>
			<td style="width:115px;">NL-15</td>
			<td style="width:251px;">Used to analyze gut fiber architecture, not necessary to have</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:217px;">Northern Eclipse</td>
			<td style="width:175px;">Empix Inc</td>
			<td style="width:115px;"></td>
			<td style="width:251px;">Imaging software</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:217px;">G-2E/C TRITC EX 528-553</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;">96312</td>
			<td style="width:251px;">Filter for specific secondary antibody</td>
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		<tr height="80">
			<td height="80" style="height:80px;width:217px;">N.A. 0.75; W.D. 0.72 mm; DIC Prism: 40xI, 40x I-C; Spring loaded</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;">MRH00400</td>
			<td style="width:251px;">Objective used for imaging</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:217px;">Simple Neurite Tracer</td>
			<td style="width:175px;">NIH Image J</td>
			<td style="width:115px;"></td>
			<td style="width:251px;"><a href="http://fiji.sc/Simple_Neurite_Tracer">http://fiji.sc/Simple_Neurite_Tracer</a></td>
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functional analysis of the larval feeding circuit in drosophila

Le circuit d&#39;alimentation sérotoninergique chez Drosophila melanogaster de larves peut être utilisé pour étudier les substrats neuronaux d&#39;une importance critique au cours de la mise au point du circuit. Utilisation de la sortie fonctionnelle du circuit, l&#39;alimentation, les changements dans l&#39;architecture neuronale du système stomatogastrique peut être visualisée. Le comportement alimentaire peut être enregistrée par l&#39;observation du taux de rétraction des crochets de la bouche, qui reçoivent l&#39;innervation du cerveau. comportement locomoteur est utilisé en tant que contrôle physiologique pour l&#39;alimentation, étant donné que les larves utilisent leurs crochets de la bouche à parcourir à travers un substrat de gélose. Les changements dans le comportement alimentaire peuvent être corrélées avec l&#39;architecture axonale des neurites innervant l&#39;intestin. Utilisation de l&#39;immunohistochimie, il est possible de visualiser et de quantifier ces modifications. Une mauvaise manipulation des larves pendant paradigmes de comportement peut modifier les données car ils sont très sensibles aux manipulations. Imagerie appropriée de l&#39;architecture des neurites innervantl&#39;intestin est critique pour la quantification précise du nombre et de la taille des varicosités ainsi que de l&#39;étendue de noeuds de branche. L&#39;analyse de la plupart des circuits de permettre seulement à la visualisation de l&#39;architecture des neurites ou des effets comportementaux, mais ce modèle permet de corréler la sortie du circuit fonctionnel avec les déficiences de l&#39;architecture neuronale.

Le circuit d&#39;alimentation sérotoninergique dans la larve de drosophile peut servir de modèle extrêmement efficace pour observer l&#39;influence de certains facteurs sur le développement du système nerveux. En quantifiant le taux d&#39;alimentation, il est possible de relier l&#39;architecture axonale du circuit d&#39;alimentation dont la sortie fonctionnelle (figure 1). Le dosage locomotrice est utilisé en tant que contrôle physiologique pour les rétractions de la bouche des crochet...

Watch this Scientific Journal Video about Functional Analysis of the Larval Feeding Circuit in Drosophila at JoVE.com

Functional Analysis of the Larval Feeding Circuit in Drosophila

Le circuit de l&#39;alimentation chez la drosophile larves melanogaster propose un modèle simple mais puissant qui permet de modifier la fréquence d&#39;alimentation pour être en corrélation avec des modifications dans les circuits neuronaux stomatogastrique. Ce circuit est composé de neurones sérotoninergiques centraux qui envoient des projections vers les crochets buccaux ainsi que l&#39;intestin antérieur.

Analyse fonctionnelle du circuit d&#39;alimentation des larves dans Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

functional-analysis-of-the-larval-feeding-circuit-in-drosophila

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JoVE Journal

Neuroscience

Functional Analysis of the Larval Feeding Circuit in Drosophila

10.2K vues.  Saint Louis University School of Medicine.  Le circuit de l'alimentation chez la drosophile larves melanogaster propose un modèle simple mais puissant qui permet de modifier la fréquence d'alimentation pour être en corrélation avec des modifications dans les circuits neuronaux stomatogastrique. Ce circuit est composé de neurones sérotoninergiques centraux qui envoient des projections vers les crochets buccaux ainsi que l'intestin antérieur. 

Vidéo: Functional Analysis of the Larval Feeding Circuit in Drosophila

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

Visualisation des larves nerfs segmentaires en 3 Rd Instar Drosophile Les préparatifs des larves

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

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Neurosciences

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Visualisation in vivo des vésicules synaptiques Dans Drosophila larves axones segmentaire

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Analyse morphologique des Drosophile Larvaires périphérique dendrites et des axones des neurones sensoriels utilisant mosaïques génétiques

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualisation des propriocepteurs dans<em&gt; Drosophila</emLes larves&gt; et pupes

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Dissection et immunohistochimique de larve, de nymphe et adulte<em&gt; Drosophila</em&gt; Rétines

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Tout le mont immunomarquage des récepteurs olfactifs neurones dans le<em&gt; Drosophila</em&gt; Antenne

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Imagerie en direct de<em&gt; Drosophila</em&gt; larves Neuroblastes

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Pourquoi Quantification Matters: Caractérisation des phénotypes au<em&gt; Drosophila</em&gt; Larvaire jonction neuromusculaire

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Enlèvement de<em&gt; Drosophila</em&gt; Muscle Tissue de Fillets larvaires pour immunofluorescence Analyse des neurones sensoriels et les cellules épidermiques

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Focal Macropatch enregistrements de courants synaptiques de la jonction neuromusculaire larves de drosophile

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...