The authors thank RIKEN for funding. We also thank Cagri Yalgin, Caroline Delandre, and Jay Parrish for discussions on genetic and immunohistochemistry protocols.

1.Preparation of reagents
<ol>
 <li>Prepare Ca++ -free HL3.1 saline28.
 <ol>
 <li>In mM: 70 NaCl, 5 KCl, 20 MgCl2 , 10 NaHCO3 , 5 HEPES, 115 sucrose, and 5 trehalose; pH 7.2. Filter sterilize and store at 4&deg;C. 
 Note: Ca++ -free solution prevents muscle contraction during dissection.</li>
 </ol>
 </li>
 <li>Make poly-L-lysine (PLL) coverslips.
 <ol>
 <li>Dissolve 100mg PLL in 4.2ml water and make 300&mu;l aliquots in Eppendorf tubes and freeze at -20&deg;C.</li>
 <li>Before coating coverslips, first thaw an aliquot, bring it up to 10ml in ddH2O and add 20&mu;l of Kodak Photo-Flo; the final PLL concentration is 0.7 mg/ml.</li>
 <li>Submerge coverslips for 30mins in PLL solution, remove and dry, then rinse briefly with water and dry. Repeat twice. Treated coverslips last approximately one month.</li>
 </ol>
 </li>
</ol>
2. Genetic crosses
<ol>
 <li>To generate MARCM clones. Here is an example cross using a pan-DA driver11,27: 
 FRT2A x hsFLP; Gal4109(2)80, UAS-mCD8::GFP; tub-Gal80 FRT2A/SM5-TM6B</li>
 <li>To generate Flp-out clones. Here is an example cross using a class IV-specific driver8: 
 ppk-Gal4 x yw, hsFLP;UAS-FRT-CD2,y+-stop-FRT-mCD8::GFP</li>
</ol>
3. Collection of embryos
<ol>
 <li>Keep Drosophila crosses in a collection bottle at 25&deg;C and collect embryos on an apple juice agar plate29 spread with a thin layer of yeast paste30,31.</li>
</ol>
4. Heat shock treatment of embryos
<ol>
 <li>Place an empty plate in opposition to the apple juice agar plate upon which the embryos have been laid. Seal around these two plates with Parafilm (Fig. 2).</li>
 <li>During heat shock, submerge the plate in the water bath and hold it down using a metal weight.</li>
 <li>For MARCM embryos
 <ol>
 <li>Collect embryos for 2h and incubate in a 10cm petri dish surrounded by moistened tissues at 25&deg;C for an additional 2h. 
 Note: Adjusting the heat shock protocol alters the frequency at which clones are generated.</li>
 <li>For a smaller number of clones, aiming for single isolated neurons, submerge and heat shock in the water bath for 1h at 38&deg;C.</li>
 <li>For a larger number of clones, heat shock for 45mins at 38&deg;C, recover at RT 30mins, then heat shock again for an additional 30mins.</li>
 </ol>
 </li>
 <li>Collect Flp-out embryos for 24h, and heat shock for 1h at 38&deg;C.</li>
</ol>
5. Screening for clones
<ol>
 <li>After heat shock, remove the lid of the water-tight arrangement and place the apple juice agar plate in a 10cm petri dish surrounded by moistened tissues. Culture the embryos and subsequent larvae at 25&deg;C until wandering 3rd instar. 
 Note: culture conditions, especially nutrition, have been shown to alter DA dendritic arbor morphology32,33. Make sure that growing larvae have access to yeast paste at all times; monitor and replenish the yeast paste as required.</li>
 <li>From this point in the protocol onwards, manipulate larvae gently using insect forceps.</li>
 <li>Briefly and gently rinse the larvae in tap water, and then place onto an agar plate. 
 Note: This step reduces background auto-fluorescence from food or dirt on the larval surface.</li>
 <li>Examine the larvae under a powerful fluorescence dissecting microscope. Identify larvae with GFP-positive neurons/cells in the body wall.</li>
</ol>
6. in vivo imaging of dendrites
<ol>
 <li>Place the larva into a depression slide glass with a small drop of 80% glycerol. Place a coverslip on slide to immobilize the larva. Ensure that no air remains between the larva and the coverslip.</li>
 <li>Gently push the coverslip to roll the larva to allow visualization of the neuron of interest. Image the mCD8::GFP-labeled dendritic arbor via confocal microscopy.</li>
</ol>
7. Larval dissection
Note before beginning: DA neuron dendrites degrade rapidly after the initiation of dissection. Dissect each individual larva in less than 5min to ensure good dendrite morphology.
<ol>
 <li>Dissect wandering 3rd instar larvae on a Sylgard plate as described previously34 with the following modifications:
 <ol>
 <li>For the imaging of DA neuron axon termini, ensure that the CNS is intact and the segmental nerves are not broken. After opening the larvae, first carefully cut the tracheal connections to the body wall and then gently remove the entire gut.</li>
 <li>If imaging the dendrites alone, remove the CNS to allow a flatter mounting.</li>
 </ol>
 </li>
</ol>
8. Fixation and blocking of larval fillets
<ol>
 <li>With the larva still pinned to the Sylgard plate, fix it in 4% PFA in PBS for 20mins at RT on a gently rotating shaker.</li>
 <li>Remove traces of larval tissues (fat body, trachea, etc.) after fixation.</li>
 <li>Wash in PBST (PBS with 0.1% Triton X-100) 3x 10mins on the Sylgard plate and shaker. If examining axon termini, keep the fillet on the Sylgard plate. (If staining dendrites it is possible to transfer the larval fillets to a 0.5 ml tube at this step34)</li>
 <li>Block the larvae for 20mins at RT in 5% normal donkey serum (NDS) in PBST on a shaker.</li>
</ol>
9. Staining of larval fillets
<ol>
 <li>Remove the blocking solution and incubate in primary antibody (in 5%NDS/PBST) overnight at 4&deg;C in a small Tupperware container surrounded by moistened tissues.</li>
 <li>Remove the tubes from 4&deg;C and incubate an additional hour at RT.</li>
 <li>Wash 6x 10mins in PBST. Add the secondary antibody (in 5%NDS/PBST), and cover to prevent fluorophore photo-bleaching34.</li>
 <li>Incubate either at RT for two hours, or overnight at 4&deg;C followed by one hour at RT.</li>
 <li>Wash the larvae 6x 10mins in PBST and proceed to mounting.</li>
</ol>
10. Mounting of larval fillets for examination of the dendrite arbor
<ol>
 <li>Mount each larval fillet as flat as possible using dissecting scissors or a scalpel to cut off the head (including the mouth hooks), and the posterior (including the spiracles)34.</li>
 <li>Place the larval fillet on the slide cuticle-side down, mount in 80% glycerol, and seal the sides of the coverslip with nail polish for a 'quick' mount34.</li>
 <li>For a permanent mounting and a clearer image
 <ol>
 <li>Place the dissected larval fillet muscle-side down onto a drop of PBS on a PLL coverslip, it will quickly adhere to the coverslip.</li>
 <li>Remove as much liquid as possible after mounting, however do not allow it to completely dry.</li>
 <li>Take the coverslip (with attached larval fillet) through an ethanol series: 35% ethanol, followed by 50%, 70%, 95%, and finally 2x 100% ethanol each for 10 minutes (the 2nd 100% EtOH solution should be changed frequently); finally, wash 2-3x 10mins in Xylene.</li>
 <li>Put a drop of DPX mountant (Distyrene Plasticizer Xylene) on a clean slide and carefully lay the coverslip fillet-side-down on top. Keep in the dark, and wait one day for the DPX to set before imaging.</li>
 </ol>
 </li>
</ol>
11. Mounting of larval fillets for examination of the axon termini
<ol>
 <li>Keep the segmental nerves running between the PNS and the CNS intact during dissection, fixation, and staining to allow the tracing of the axons from peripheral sensory neuron cell body to the VNC.</li>
 <li>Mount each larval fillet cuticle-side down in 80% glycerol in a depression slide. Trace the axons from the DA cell body to the VNC under the confocal microscope. Note: PNS neurons in each body wall segment project to the cognate segment of the VNC.</li>
</ol>
12. Representative Results:
Representative results are shown in figures 3-5. Fig. 3 shows the entire arbor of a class IV da neuron, captured in vivo under the confocal microscope. Fig. 4 shows a close up of part of the dendrite arbor of an immunohistochemically labeled class III neuron that has been correctly fixed to preserve morphology. The associated inset shows the degradation that can occur after an unsuccessful dissection and fixation (both stained with anti-GFP antibody). Fig. 5 shows a single class IV axon terminus in the CNS (anti-GFP, green); all class IV termini are co-stained with anti-CD2 (magenta).
<img alt="Figure 1" src="/files/ftp_upload/3111/3111fig1.jpg" /> 
Figure 1 Protocol overview. a) Collect and then heat-shock embryos. b) Select a larva with a GFP-positive DA neuron clone, and then image the GFP-labeled dendrite arbor in the live larva. c) Dissect the larva, and then immunohistochemically stain the fillet. d) Mount the stained fillet, and then image the dendritic arbor and axonal projections of the DA neuron clones.
<img alt="Figure 2" src="/files/ftp_upload/3111/3111fig2.jpg" /> 
Figure 2 Preparation of the apple juice agar plate for heat shock. Take the plate (white arrow, a-b), add a second petri dish on top (red arrow b) and seal around with Parafilm (blue arrow, b).
<img alt="Figure 3" src="/files/ftp_upload/3111/3111fig3.jpg" /> 
Figure 3 in vivo image of the dendrite arbor of a mCD8::GFP-labeled MARCM clone (genotype as in 2.1) representing a class IV (v'ada) neuron of a 3rd instar larva. Scale bar is 50&mu;m.
<img alt="Figure 4" src="/files/ftp_upload/3111/3111fig4.jpg" /> 
Figure 4 mCD8::GFP-labeled MARCM clone of a class III neuron (ddaA) stained with anti-GFP antibody, showing good morphology after successful dissection and fixation (green). The inset shows dendrite degradation (white arrows) occurring after unsuccessful dissection and fixation (magenta). Scale bar is 25&mu;m.
<img alt="Figure 5" src="/files/ftp_upload/3111/3111fig5.jpg" /> 
Figure 5 The 3rd instar larval VNC. A Flp-out clone of a single class VI (vdaB) axon terminus (genotype as in 2.2) is detected using anti-GFP antibody (green). Anti-CD2 labels all class IV termini (magenta). Scale bar is 25&mu;m.

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li><li>Landgraf, M., Sanchez-Soriano, N., Technau, G. M., Urban, J., Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+the+Drosophila+neuropile:+a+strategy+for+the+standardised+characterisation+of+genetically+amenable+neurites.">Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites.</a> Dev Biol. 260, 207-225 (2003).</li></ol>

No conflicts of interest declared.

The Drosophila larval DA neuron model provides one excellent genetic system to investigate mechanisms that control neuron morphology and circuit formation. MARCM is generally used for labeling and for generating mutant DA neuron clones. For MARCM we use either a pan-neural (e.g. Gal4c155) or DA neuron-specific driver. Using a pan-neural driver it is possible to directly use several stocks widely available from public stock centers. However using a DA-neuron specific driver can be adv...

<table><tbody><tr><td>SZX16 fluorescence dissection microscope (with GFPHQ filter)</td><td>Olympus Corporation</td><td>SZX16</td><td></td></tr><tr><td>Live Insect Forceps</td><td>Fine Science Tools</td><td>26030-10</td><td></td></tr><tr><td>26mm x 76mm depression slide glass</td><td>Toshinriko Co.</td><td>T8-R004</td><td></td></tr><tr><td>Sylgard 184 (or Silpot 184)</td><td>Dow Corning</td><td>3097358-1004</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P-1524</td><td>This product has proven most effective</td></tr><tr><td>DPX mounting medium</td><td>Sigma-Aldrich</td><td>44581</td><td></td></tr><tr><td>Rabbit anti-GFP</td><td>Invitrogen</td><td>A-11122</td><td>Dilution 1:500</td></tr><tr><td>Rat anti-CD8</td><td>Caltag</td><td>5H10</td><td>Dilution 1:200</td></tr><tr><td>Mouse anti-CD2</td><td>AbD Serotec</td><td>MCA443R</td><td>Dilution 1:700</td></tr><tr><td>Mouse anti-Fasciclin2</td><td>DSHB</td><td>1D4</td><td>Dilution 1:10</td></tr></tbody></table>

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).

Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

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.

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 ...

morphological-analysis-drosophila-larval-peripheral-sensory-neuron

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.2K Views.  RIKEN Brain Science Institute. 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.

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

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Developmental Biology

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

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.

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

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

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

Summary

Explore More Videos

Chapters in this video

Laser Capture Microdissection of Drosophila Peripheral Neurons

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

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

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila