We thank Gary Laevsky for helpful discussions on microscopy. This work was funded by NIH grants R01GM061107 and R01GM067758 to E.R.G.

Note: The procedure for muscle removal (Figure 1) is a modification of previously described methods for preparing larval fillets. The steps that precede and follow muscle removal are outlined briefly and the reader is referred to previous work 10, 11 for more detailed descriptions.
1. Dissect Larva in Cold Saline
<ol>
	<li>Prepare a working dilution of cold HL3.1 saline15 or cold Ca2+-free HL3.1 saline11 (Table 1). Place the larva in a silicone elastomer dish with just enough cold saline to cover the bottom of the dish. 
	NOTE: See Discussion regarding the choice of saline.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x HL3.1 saline (pH 7.2)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 5 mM HEPES</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 70 mM NaCl</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 5 mM KCl</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 1.5 mM CaCl2&#160;&#160; (omit for Ca2+-free saline)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 4 mM MgCl2</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 10 mM NaHCO3</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 5 mM trehalose</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 115 mM sucrose</td>
		</tr>
		<tr>
			<td></td>
		</tr>
		<tr>
			<td>Filter sterilize and store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Note: Composition mimics insect hemolymph</td>
		</tr>
	</tbody>
</table>
Table 1. Composition of Cold Saline.
<ol start="2">
	<li>Position the larva with the ventral side up. The ventral surface of the larva can be identified by the abdominal dentical belts and the dorsal side by the primary larval tracheal tubes running from anterior to posterior. Stretch the larva in the anterior-posterior direction and pin the head and tail to a silicone elastomer dissecting dish using insect pins. Use fine dissecting scissors to cut along the ventral midline, beginning at one end and progressing toward the other. 
	NOTE: This orientation preserves the commonly studied dorsal cluster of da neurons.</li>
	<li>After the larva is cut open, pin the four corners to the dissecting dish as though opening a book. Use forceps to grab and remove internal organs including the CNS, gut, and trachea. Adjust insect pins so that the fillet is taut but not maximally stretched. 
	NOTE: Muscles are anchored to the body wall at segmental boundaries. Although muscles cover most of the body wall, they are absent in a narrow region near the dorsal midline.</li>
</ol>
2. Muscle Removal
<ol>
	<li>Locate the dorsal midline of the larva, where muscle tissue is absent. Position a single forceps prong such that it can be inserted underneath the muscle tissue in the flattest possible orientation.</li>
	<li>Starting at the dorsal midline, near the anterior boundary of the segment, carefully slide the forceps prong between the muscle and epidermis, taking care to minimize contact between the forceps and epidermis.</li>
	<li>Pull the forceps upwards to break the attachment of the muscle to the body wall at one anchor point. Repeat this process for the remaining hemi-segment(s) of interest. 
	NOTE: This protocol optimizes preservation of the posterior of each da neuron dendrite field. To preserve the anterior part of the dendrite field, it is best to insert the forceps prong at the posterior end of each segment.</li>
	<li>Re-adjust insect pins such that the larval fillet is maximally stretched in all directions.</li>
	<li>Fix the fillet while it is still pinned to the dissecting dish using cold, freshly prepared 4% formaldehyde in PBS for 25 min.</li>
	<li>Rinse 5 times in PBS.</li>
	<li>Use forceps to carefully pull away muscle tissue from the remaining anchor points, taking care to minimize contact with the epidermis.</li>
	<li>Unpin and remove the fillet from the dissecting dish to a 1.5 ml microcentrifuge tube. Perform all subsequent washing, blocking, and immunofluorescence staining steps, as previously described11.</li>
</ol>
3. Mount the Larval Fillet
<ol>
	<li>First remove the head and tail using fine dissecting scissors in order to make the sample as flat as possible. Mount the fillet on a coverslip in antifade mountant with the inner surface of the fillet facing the coverslip.</li>
	<li>Place a microscope slide on the coverslip and press gently to disperse the mounting medium. Flip the microscope slide over and seal the coverslip on the slide using nail polish. Slides can be stored at -20 &#176;C for at least one month.</li>
</ol>

<ol><li>Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Branching+out%3A+mechanisms+of+dendritic+arborization%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">Branching out: mechanisms of dendritic arborization.</a> Nat Rev Neurosci. 11 (5), 316-328 (2010).</li>
<li>Corty, M. M., Matthews, B. J., Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecules+and+mechanisms+of+dendrite+development+in+Drosophila%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Molecules and mechanisms of dendrite development in Drosophila.</a> Development. 136 (7), 1049-1061 (2009).</li>
<li>Parrish, J. Z., Emoto, K., Kim, M. D., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+that+regulate+establishment%2C+maintenance%2C+and+remodeling+of+dendritic+fields%5BTitle%5D)+AND+%22Annu+Rev+Neurosci%22%5BJournal%5D)">Mechanisms that regulate establishment, maintenance, and remodeling of dendritic fields.</a> Annu Rev Neurosci. 30, 399-423 (2007).</li>
<li>Kim, M. E., Shrestha, B. R., Blazeski, R., Mason, C. A., Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrins+establish+dendrite-substrate+relationships+that+promote+dendritic+self-avoidance+and+patterning+in+Drosophila+sensory+neurons%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Integrins establish dendrite-substrate relationships that promote dendritic self-avoidance and patterning in Drosophila sensory neurons.</a> Neuron. 73 (1), 79-91 (2012).</li>
<li>Han, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrins+regulate+repulsion-mediated+dendritic+patterning+of+Drosophila+sensory+neurons+by+restricting+dendrites+in+a+2D+space%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Integrins regulate repulsion-mediated dendritic patterning of Drosophila sensory neurons by restricting dendrites in a 2D space.</a> Neuron. 73 (1), 64-78 (2012).</li>
<li>Parrish, J. Z., Xu, P., Kim, C. C., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+microRNA+bantam+functions+in+epithelial+cells+to+regulate+scaling+growth+of+dendrite+arbors+in+Drosophila+sensory+neurons%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The microRNA bantam functions in epithelial cells to regulate scaling growth of dendrite arbors in Drosophila sensory neurons.</a> Neuron. 63 (6), 788-802 (2009).</li>
<li>Jiang, N., Soba, P., Parker, E., Kim, C. C., Parrish, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+microRNA+bantam+regulates+a+developmental+transition+in+epithelial+cells+that+restricts+sensory+dendrite+growth%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The microRNA bantam regulates a developmental transition in epithelial cells that restricts sensory dendrite growth.</a> Development. 141 (13), 2657-2668 (2014).</li>
<li>Meltzer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermis-derived+Semaphorin+promotes+dendrite+self-avoidance+by+regulating+dendrite-substrate+adhesion+in+Drosophila+sensory+neurons%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Epidermis-derived Semaphorin promotes dendrite self-avoidance by regulating dendrite-substrate adhesion in Drosophila sensory neurons.</a> Neuron. 89 (4), 741-755 (2016).</li>
<li>Han, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermal+cells+are+the+primary+phagocytes+in+the+fragmentation+and+clearance+of+degenerating+dendrites+in+Drosophila%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila.</a> Neuron. 81 (3), 544-560 (2014).</li>
<li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+larval+NMJ+dissection%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li>
<li>Karim, M. R., Moore, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological+analysis+of+Drosophila+larval+peripheral+sensory+neuron+dendrites+and+axons+using+genetic+mosaics%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics.</a> J Vis Exp. (57), e3111(2011).</li>
<li>Gustafsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy%5BTitle%5D)+AND+%22J+Microscopy%22%5BJournal%5D)">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> J Microscopy. 198 (2), 82-87 (2000).</li>
<li>Ashdown, G. W., Cope, A., Wiseman, P. W., Owen, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+flow+quantified+beyond+the+diffraction+limit+by+spatiotemporal+image+correlation+of+structured+illumination+microscopy+data%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Molecular flow quantified beyond the diffraction limit by spatiotemporal image correlation of structured illumination microscopy data.</a> Biophys J. 107 (9), L21-L23 (2014).</li>
<li>Zhang, W., Yan, Z., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sound+response+mediated+by+the+TRP+channels+NOMPC%2C+NANCHUNG%2C+and+INACTIVE+in+chordotonal+organs+of+Drosophila+larvae%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Sound response mediated by the TRP channels NOMPC, NANCHUNG, and INACTIVE in chordotonal organs of Drosophila larvae.</a> PNAS. 10 (33), 13612-13617 (2013).</li>
<li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modified+minimal+hemolymph-like+solution%2C+HL3.1%2C+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae%5BTitle%5D)+AND+%22J+Neurogenet%22%5BJournal%5D)">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18 (2), 377-402 (2004).</li>
<li>Babcock, D. T., Landry, C., Galko, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytokine+signaling+mediates+UV-induced+nociceptive+sensitization+in+Drosophila+larvae%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae.</a> Curr Biol. 19 (10), 799-806 (2009).</li>
</ol>

The authors have nothing to disclose.

Here a protocol is described for manual removal of muscle tissue from Drosophila larval fillets. This protocol modifies previously described larval dissection techniques10,11. After the larva is dissected in a silicone elastomer dish, the dorsal midline is located. A single forceps prong, in its flattest possible orientation, is carefully inserted between the muscle tissue and the epidermis, near the dorsal midline. The forceps are gently pulled upwards to separate muscle tissue from one anchor point ...

Drosophila larval dendritic arborization (da) neurons provide a valuable model for studying neuronal development due to their amenability to genetic manipulation and the ease with which they can be imaged. These sensory neurons have been instrumental in the identification of numerous pathways that control dendrite morphogenesis1-3.
Four classes of da neurons (class I - IV) innervate the larval epidermis. These neurons lie between the basement membrane and the epidermis, with their dendrites forming largely two-dimensional arrays4,5. Of the four classes, class IV da neurons have the most highly branched arbors and, like sensory neurons of other animals, the elaboration of these arbors requires intrinsic factors as well as cues from neighboring tissues, particularly the epidermis, for their development6-9.
Studies to determine how such neuronal and extra-neuronal factors control dendrite morphogenesis benefit from the ability to detect protein expression in situ by immunofluorescence. The outer cuticle of the larva is impenetrable to antibodies, but this impediment is easily overcome by the preparation of larval fillets through well-established dissection methods10,11. However, the body wall muscle tissue that lies just interior to the basement membrane presents several challenges towards visualization of da neurons and epidermal cells. First, the muscle tissue, which lines most of the body wall, greatly obscures fluorescent signals emanating from neuronal or epidermal tissue. This substantially reduces the signal to noise ratio in the sample. Second, many relevant proteins may be expressed in the muscle tissue as well as in the neurons or epidermis. This muscle-derived fluorescence signal is likely to further obscure detection of a fluorescence signal from the neuron or epidermis. Finally, advances in microscopy technologies now permit imaging of samples at sub-diffraction resolution and may be especially helpful in discerning the localization of proteins that are expressed in neurons and surrounding epidermal cells12,13. However, imaging via super-resolution microscopy benefits from a strong signal to noise ratio and close proximity of the sample to the coverslip. In addition to reducing the signal to noise ratio, the larval body wall muscle distances da neurons from the coverslip, thereby limiting the improved image resolution that can be achieved with super-resolution microscopy methods. Besides challenges for immunofluorescence analysis, muscle tissue presents a barrier to electrophysiological recording from sensory neurons in the larval body wall. Its removal therefore benefits neurophysiological manipulation of sensory neurons14.
Here a method for manual removal of Drosophila larval muscle tissue is described. We demonstrate that our protocol permits immunofluorescence imaging of proteins that are otherwise obscured by muscle tissue, improves the signal to noise ratio for visualization of class IV da neurons, and enables the use of super-resolution microscopy to better discriminate spatial relationships of proteins and cellular structures in da neurons and the epidermis.

<table><tbody><tr><td>Dumont #5 tweezers</td><td>Electron Microscopy Sciences</td><td>72701-D</td><td></td></tr><tr><td>Micro Scissors, 8 cm, straight, 5 mm blades, 0.1 mm tips</td><td>World Precision Instruments</td><td>14003</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>Dow Corning</td><td>3097358-1004</td><td>for dissecting plates</td></tr><tr><td>Austerlitz insect pins, 0.1 mm</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Fostec 8375 light source</td><td>Artisan Technology Group</td><td>62792-4</td><td></td></tr><tr><td>Zeiss Stemi 2000</td><td>Carl Zeiss Microscopy</td><td></td><td></td></tr><tr><td>Vectashield antifade mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td>for confocal microscopy</td></tr><tr><td>Prolong Diamond antifade mountant</td><td>Life Technologies</td><td>P36970</td><td>for structured illumination microscopy</td></tr><tr><td>Micro cover glass, 22 x 22 mm, No. 1.5</td><td>VWR</td><td>48366-227</td><td></td></tr><tr><td>Superfrost Plus microscope slides, 25 x 75 x 1.0 mm</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>Mouse anti-Coracle antibody</td><td>Developmental Studies Hybridoma Bank</td><td>C615.16</td><td>supernatant, dilute 1:50</td></tr><tr><td>Mouse anti-Discs large antibody</td><td>Developmental Studies Hybridoma Bank</td><td>4F3</td><td>supernatant, dilute 1:50</td></tr><tr><td>Rabbit anti-dsRed antibody</td><td>Clontech</td><td>632496</td><td>dilute 1:1,000</td></tr><tr><td>Goat anti-rabbit antibody, Alexa Fluor 568 conjugated</td><td>ThermoFisher Scientific</td><td>A-11011</td><td>dilute 1:1,000</td></tr><tr><td>Goat anti-mouse antibody, Alexa Fluor 488 conjugated</td><td>ThermoFisher Scientific</td><td>A-11001</td><td>dilute 1:500</td></tr></tbody></table>

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.

We demonstrate the utility of the muscle removal procedure for improving signal to noise ratio in immunofluorescence experiments to co-visualize the septate junction proteins Coracle (Cora) and Discs-large (Dlg) together with class IV da neurons labeled with the membrane marker CD4-tdTomato.
Cora has been previously used to identify tracts where da neuron dendrites are enclosed by epidermal cells and is one of many identified epidermal factors that have been st...

Watch this Scientific Journal Video about Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells at JoVE.com

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.

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 ...

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9.0K Views.  Princeton University. 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.

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

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

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.

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

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

Genetics

Drosophila Larval NMJ Dissection

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

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

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

Biology

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Viewing Microtubule Networks in Drosophila Neuromuscular Junctions and Muscle Cells

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

Single-Neuron Ablation in Drosophila for Studying Nervous System Adaptation

Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. Using this non-invasive method, the developing nervous system can be manipulated in a cell-specific manner. Disrupting the development of individual neurons in a network can be used to study how the nervous system can compensate for the loss of synaptic input. Individual neurons were specifically ablated in the giant fiber system of Drosophila, with a focus on two neurons: the presynaptic giant fiber (GF) and the postsynaptic tergotrochanteral motor neuron (TTMn). The GF synapses with the ipsilateral TTMn, which is crucial to the escape response. Ablating one of the GFs in the 3rd instar brain, just after the GF starts axonal growth, permanently removes the cell during the development of the CNS. The remaining GF reacts to the absent neighbor and forms an ectopic synaptic terminal to the contralateral TTMn. This atypical, bilaterally symmetric terminal innervates both TTMns, as demonstrated by dye coupling, and drives both motor neurons, as demonstrated by electrophysiological assays. In summary, the ablation of a single interneuron demonstrates synaptic competition between a bilateral pair of neurons that can compensate for the loss of one neuron and restore normal responses to the escape circuit.

Author Spotlight: Investigating the Mechanisms of Neural Circuit Assembly and Synapse Formation in Drosophila

This protocol demonstrates the laser cell ablation of individual neurons in intact Drosophila larvae. The method enables the study of the effect of reducing competition between neurons in the developing nervous system.

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. ...

Drosophila Late Pupa Indirect Flight Muscle (IFM) Dissection: A Method for High-Throughput Tissue Collection

Source: Kao, S. Y., et al. <a href="https://www.jove.com/video/60309/" target="_blank">Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches</a>. J. Vis. Exp. (2019).
The Drosophila flight muscles are used as a model system for muscle development and physiology. This video describes the flight musculature of adults and highlights a protocol to dissect developing indirect flight muscles that are suitable for RNA sequencing from pupae.

Source: Kao, S. Y., et al. Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches. J. Vis. Exp. (2019).
The Drosophila flight muscles ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Pupa

Larval Fillet Preparation: A Method to Visualize Intact Sensory Neurons and Associated Epidermal Cells

Source: Tenenbaum, C. M., Gavis, E. R. <a href="https://www.jove.com/video/54670/" target="_blank">Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells</a>. J. Vis. Exp. (2016).
This video describes how to prepare larval fillets used to visualize sensory dendritic arborization (da) neurons and their associated epidermal cells. The featured protocol shows in detail how to remove muscle tissue from the body wall while preserving the cells&#39; morphology, which improves immunostaining and visualization of the otherwise obscured da neurons and epidermal cells.

Source: Tenenbaum, C. M., Gavis, E. R. Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and ...

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

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Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

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Drosophila Late Pupa Indirect Flight Muscle (IFM) Dissection: A Method for High-Throughput Tissue Collection

Larval Fillet Preparation: A Method to Visualize Intact Sensory Neurons and Associated Epidermal Cells