Name: retrograde tracing of drosophila embryonic motor neurons using lipophilic fluorescent dyes
Uploaded: 2020-01-12T16:00:00
Description: Watch this Scientific Journal Video about Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes at JoVE.com

We thank members of the Kamiyama Lab for comments on the manuscript. This work was supported by an NIH R01 NS107558 (to M.I., K.B., and D.K.).

1. Equipment and Supplies
<ol>
	<li>Materials for collecting embryos and training adults to lay eggs
	<ol>
		<li>Prepare the filtration apparatus by severing a 50 mL tube and cutting open a hole in the cap to set a mesh filter with pores of 100 &#181;m (Table of Materials) in between the tube and the cap. 
		NOTE: Alternatively, cell strainers with pores of 100 &#181;m (Table of Materials) can be used for the filtration step of embryo collection.</li>
		<li>Make agar plates with g...

<ol>
	<li>Arzan Zarin, A., Labrador, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+axon+guidance+in+Drosophila.">Motor axon guidance in Drosophila.</a> Seminars in Cell and Developmental Biology. 85, 36-47 (2019).</li><li>Nose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neuromuscular+specificity+in+Drosophila:+novel+mechanisms+revealed+by+new+technologies.">Generation of neuromuscular specificity in Drosophila: novel mechanisms revealed by new technologies.</a> Frontiers in Molecular Neuroscience. 5, 62 (2012).</li><li>Kim, M. D., Wen, Y., Jan, Y. 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A., Chiba, A., Umemori, H., Hortsch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Adhesion+Molecules+at+the+Drosophila+Neuromuscular+Junction.">Cell Adhesion Molecules at the Drosophila Neuromuscular Junction.</a> The Sticky Synapse: Cell Adhesion Molecules and Their Role in Synapse Formation and Maintenance. , 11-37 (2009).</li><li>Ritzenthaler, S., Suzuki, E., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+filopodia+in+muscle+cells+interact+with+innervating+motoneuron+axons.">Postsynaptic filopodia in muscle cells interact with innervating motoneuron axons.</a> Nature Neuroscience. 3 (10), 1012-1017 (2000).</li><li>Kohsaka, H., Takasu, E., Nose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+induction+of+postsynaptic+molecular+assembly+by+the+cell+adhesion+molecule+Fasciclin2.">In vivo induction of postsynaptic molecular assembly by the cell adhesion molecule Fasciclin2.</a> Journal of Cell Biology. 179 (6), 1289-1300 (2007).</li><li>Kohsaka, H., Nose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+recognition+at+the+tips+of+postsynaptic+filopodia:+accumulation+and+function+of+Capricious.">Target recognition at the tips of postsynaptic filopodia: accumulation and function of Capricious.</a> Development. 136 (7), 1127-1135 (2009).</li><li>Landgraf, M., Bossing, T., Technau, G. M., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin,+location,+and+projections+of+the+embryonic+abdominal+motorneurons+of+Drosophila.">The origin, location, and projections of the embryonic abdominal motorneurons of Drosophila.</a> Journal of Neuroscience. 17 (24), 9642-9655 (1997).</li><li>Kamiyama, D., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+activation+patterns+of+Cdc42+GTPase+within+Drosophila+embryos.">Endogenous activation patterns of Cdc42 GTPase within Drosophila embryos.</a> Science. 324 (5932), 1338-1340 (2009).</li><li>Furrer, M. P., Vasenkova, I., Kamiyama, D., Rosado, Y., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slit+and+Robo+control+the+development+of+dendrites+in+Drosophila+CNS.">Slit and Robo control the development of dendrites in Drosophila CNS.</a> Development. 134 (21), 3795-3804 (2007).</li><li>Kamiyama, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+Dendritogenesis+Site+in+Drosophila+aCC+Motoneuron+by+Membrane+Enrichment+of+Pak1+through+Dscam1.">Specification of Dendritogenesis Site in Drosophila aCC Motoneuron by Membrane Enrichment of Pak1 through Dscam1.</a> Developmental Cell. 35 (1), 93-106 (2015).</li><li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The embryonic development of Drosophila melanogaster. , (1985).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Ringer's+solution.">Drosophila Ringer's solution.</a> Cold Spring Harbor Protocols. 2007 (4), (2007).</li><li>Rickert, C., Kunz, T., Harris, K. -. L., Whitington, P., Technau, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+of+single+cells+in+the+central+nervous+system+of+Drosophila+melanogaster.">Labeling of single cells in the central nervous system of Drosophila melanogaster.</a> Journal of Visualized Experiments. (73), e50150 (2013).</li><li>Fujioka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Even-skipped,+acting+as+a+repressor,+regulates+axonal+projections+in+Drosophila.">Even-skipped, acting as a repressor, regulates axonal projections in Drosophila.</a> Development. 130 (22), 5385-5400 (2003).</li><li>Sink, H., Rehm, E. J., Richstone, L., Bulls, Y. M., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=sidestep+encodes+a+target-derived+attractant+essential+for+motor+axon+guidance+in+Drosophila.">sidestep encodes a target-derived attractant essential for motor axon guidance in Drosophila.</a> Cell. 105 (1), 57-67 (2001).</li><li>Furrer, M. P., Kim, S., Wolf, B., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robo+and+Frazzled/DCC+mediate+dendritic+guidance+at+the+CNS+midline.">Robo and Frazzled/DCC mediate dendritic guidance at the CNS midline.</a> Nature Neuroscience. 6 (3), 223-230 (2003).</li><li>Landgraf, M., Jeffrey, V., Fujioka, M., Jaynes, J. B., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origins+of+a+motor+system:+motor+dendrites+form+a+myotopic+map+in+Drosophila.">Embryonic origins of a motor system: motor dendrites form a myotopic map in Drosophila.</a> PLoS Biology. 1 (2), 41 (2003).</li></ol>

The use of dye labeling for studying neuronal morphology has several advantages over genetic cell-labeling techniques. The dye labeling technique can minimize the amount of time needed for labeling and imaging the morphologies of motor neurons. The dye labeling process is quite fast as it takes less than 2 h and enables us to define the outline of neuronal projections. As an alternative, one can visualize the aCC motor neuron by choosing a GAL4 line that expresses the yeast GAL4 transcription factor in aCC, and crossing ...

The embryonic motor neuron system of Drosophila offers a powerful experimental model to analyze the mechanisms underlying the development of the central nervous system (CNS)1,2,3. The motor neuron system is amenable to biochemical, genetic, imaging, and electrophysiological techniques. Using the techniques, genetic manipulations and functional analyses can be carried out at the level of single motor neurons2,4,5,6.
During early development of the nervous system, neuroblasts divide and generate a large number of glia and neurons. The spatiotemporal relationship between the delamination and the gene expression profile of neuroblasts has been previously investigated in detail7,8,9. In the case of the motor neuron system, the formation of embryonic neuromuscular junction (NMJ) has been extensively studied using the aCC (anterior corner cell), RP2 (raw prawn 2), and RP5 motor neurons2,10. For instance, when the RP5 motor neuron forms a nascent synaptic junction, the pre-synaptic and post-synaptic filopodia are intermingled11,12,13. Such direct cellular communication is vital to initiate the NMJ formation. Contrary to what we know about the peripheral nerve branches, our knowledge of how motor dendrites initiate synaptic connectivity within the CNS is still primitive.
In this report, we present a technique that allows retrograde labeling of motor neurons in embryos by means of micropipette-mediated delivery of lipophilic dyes. This technique enables us to trace the 38 motor neurons innervating each of the 30 body wall muscles in a hemi-segment at 15 h after egg laying (AEL)14. By using this technique, our group has thoroughly investigated numerous gain-of-function/loss-of-function alleles15,16,17. We have recently unraveled the molecular mechanisms that drive initiation of motor dendrite connectivity and demonstrated that a Dscam1-Dock-Pak interaction defines the site of dendrite outgrowth in the aCC motor neuron17. In general, this technique is adaptable for the phenotypic analysis of any embryonic motor neurons in wild type or mutant strains, enhancing our ability to provide new insights into the functional design of the Drosophila nervous system.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x objective lens</td>
			<td>Nikon</td>
			<td></td>
			<td>Plan</td>
		</tr>
		<tr>
			<td>40x water-immersion lens</td>
			<td>Nikon</td>
			<td></td>
			<td>NIR Apo</td>
		</tr>
		<tr>
			<td>Capillary tubing</td>
			<td>Frederick Haer&#38;Co</td>
			<td>27-31-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Andor</td>
			<td>N/A</td>
			<td>Dragonfly Spinning disk confocal unit</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Corning</td>
			<td></td>
			<td>22x22 mm Square #1</td>
		</tr>
		<tr>
			<td>DiD</td>
			<td>ThermoFisher</td>
			<td>V22886</td>
			<td></td>
		</tr>
		<tr>
			<td>DiI</td>
			<td>ThermoFisher</td>
			<td>V22888</td>
			<td></td>
		</tr>
		<tr>
			<td>DiO</td>
			<td>ThermoFisher</td>
			<td>V22887</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>SMZ-U</td>
		</tr>
		<tr>
			<td>Double Sided Tape</td>
			<td>Scotch</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning High-Vacuum Grease</td>
			<td>Fisher Sci.</td>
			<td>14-635-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg collection cage</td>
			<td>FlyStuff</td>
			<td>59-100</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoJet 5247</td>
			<td>Eppendorf</td>
			<td>discontinued</td>
			<td>FemtoJet 4i (Cat No. 5252000021)</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette beveler</td>
			<td>Sutter</td>
			<td>BV-10-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Narishige</td>
			<td>PC-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutri-Fly Grape Agar Powder Premix Packets</td>
			<td>FlyStuff</td>
			<td>47-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Net Filter</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% Solution, EM grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Any EM grades</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Roche</td>
			<td>11666789001</td>
			<td>Sold on sigmaaldrich, boxed 10x solution</td>
		</tr>
		<tr>
			<td>Photo-Flo 200</td>
			<td>Kodak</td>
			<td>146 4510</td>
			<td>Wetting agent</td>
		</tr>
		<tr>
			<td>Upright fluorescence microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>Eclipse Ci with a LED light source</td>
		</tr>
		<tr>
			<td>Vinyl Electrical Tape</td>
			<td>Scotch</td>
			<td>6143</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Cell Strainers</td>
			<td>VWR</td>
			<td>10199-659</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast</td>
			<td>FlyStuff</td>
			<td>62-103</td>
			<td>Active dry yeast (RED STAR)</td>
		</tr>
	</tbody>
</table>

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.

A representative image of the aCC and RP3 motor neurons is shown in Figure 3C to demonstrate the multicolor labeling of motor neurons at 15 h AEL. Their dendritic morphologies are largely invariant between embryos. The staining pattern obtained with anti-HRP antibody is shown in gray. A small droplet of DiO or DiD was deposited on the NMJ of muscle 1 or 6/7, respectively. Figure 4 demonstrates the capability to quantitatively measure the phenoty...

Watch this Scientific Journal Video about Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes at JoVE.com

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.

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 ...

The tracing technique described in this protocol helps us study neuronal morphogenesis and snap the connectivity in the motor neuron system of Drosophila. The lipophilic dye can rapidly diffuse to every part of the cellular membrane with an extremely high density. This is why our protocol efficiently resolves to find structures of a labeled neuron.If an axon terminal is accessible with an injection micropipette, this technique could be applied to any neuron in the larval and adult stages of flies or in other organisms. If you have no experience dissecting or using a microinjector precise the operation on the specimen, can be challenging. Understanding the anatomy of the embryo, will help with guidance to proper portioning of tools to carry out dissection or a dinjection.To start, prepare and layout all embryo collecting materials. Prepare the filtration apparatus by severing a 50 milliliter tube, and cutting open a hole in the cap. Then set a mesh filter with 100 micrometer pores in between the tube and the cap.Prepare the dye injection micropipette and dissection needle from the same capillary tubing with an inner diameter of 1.2 millimeters. Pull the tubing with a micropipette, puller at 7%from 170 volt maximum output to create a sharp needle with a taper of about 0.4 centimeters in length. To prepare the dye injection micropipette, soak the grinder with a wetting agent and place the needle on the micropipette clamp at 25 to 30 degrees.Then lower the tip onto two thirds of the radius out from the center of the beveling surface. Grind the needle while a syringe with tubing pushes air into it. Mark the micropipette with a fine tip permanent marker to indicate the position of the opening at the tip after beveling because it can be challenging to locate the narrow opening that is formed at an angle.Allow the flies to lay eggs overnight at room temperature and collect the pipe with the eggs in the morning. To collect the embryos, dechlorinate the eggs on the plate with 50%bleach for five minutes. Once the chlorions have cleared, pour the contents of the plate through the filtration apparatus or cell strainer to isolate the embryos.Use a squeeze bottle of water to dilute the bleach left on the plate and gather as many embryos as possible by decanting the mixture into the filter. Wash the embryos three to four times with water until the bleach odor dissipates. Remove the filter from the apparatus and wash them onto another clean plate with water.Then decant the water from the new plate that the embryos are on. Use fine forceps to select five to 10 embryos at 15 hours after egg laying and place them on double sided tape with the dorsal side facing up. Add insect ringer saline to the dissection pool to protect the embryos from desiccation.Use a glass needle under a dissection microscope to drag the embryo out from the middling membrane from the tape onto the glass, taking care not to damage the interior tissues of the embryo. Then cut through the mid line of a single embryo at it's surface from its posterior to anterior end. Flip the epithelial tissues from the center and attach the epidermal edge onto the surface of the glass slide.Use a tube connected needle with a tip opening of about 300 micrometers to aspirate or blow air to remove the dorsal longitudinal tracheal trunks as well as any remaining guts. Use 4%PFA and PBS to fix the embryos for five minutes at room temperature on an orbital shaker. Then wash them three times with PBS.Stain the embryos with one microliter of anti horseradish peroxidase antibody conjugated with Cyanine3 dye and 200 microliters of PBS for one hour on the orbital shaker. And repeat the washes in PBS. To fill the injection micropipette, place it into the capillary holder.Next place the dye slide onto the stage and use the micromanipulator to position it over the slide. Then adjust the stage to place the micropipette onto the dye. Collect the dye in the micropipette by setting the injection pressure to between 200 and 500 hectopascal, the injection time between 0.1 and 0.5 seconds and the compensation pressure to zero for five minutes.Once the dye has been collected, remove the dye slide and place the sample onto the microscope stage. Then increase the compensation pressure to a range of 30 to 60 hectopascal and lower the micropipette into the sample. Use the 10 times objective lens to locate the embryo and align the micropipette with the embryo.Change the objective lens to a water immersion 40 times lens. And submerge the lens into the PBS. Use fluorescence microscopy to check the neuronal morphology marked by anti-HRP Cy3 and determine the injection site.When the embryo is in focus change the position of the micropipette to make gentle contact with the tip of the axon of interest. Then use bright field microscopy during the injection to see the dye droplet. Drop the dye in the right abdominal hemisegment at the neuromuscular junction of aCC or RP3 with either DiD or DiO.Use the hand control to release the dye and remove the micropipette. Then move on to the next injection site. Incubate the sample for one hour at room temperature on an orbital shaker prior to imaging.Using fine forceps, remove the tapes from the glass slide. To mount the sample, prepare a cover slip with a small amount of vacuum grease at the four corners. Carefully placed it on the sample, making sure to avoid air bubbles.Push down the cover slip to adjust the space from the sample for allowing working distance between the objective lens and the slide. Remove any excess PBS with task wipes. Completely seal the edges of the cover slip with nail polish.Image at 10 times and 100 times magnification with a confocal microscope and process the images, with the imageJ software. This protocol was used to retrograde label the aCC motor neuron innervating muscle one and the RP3 motor neuron innervating muscles six and seven. Dendritic branches from the ACC and RP3 motor neurons show extensive overlap.Both neurons are bipolar, establishing two different populations of dendrites. To demonstrate the quantitative capabilities of this method, the total number of dendrite tips was counted in wild type and Dscam1 mutants. The ipsilateral dendrites of aCC are shown for the wild type, but few ipsilateral dendrites were observed for the Dscam1 mutant.When attempting this procedure, it is important to remember that the size of the dye droplet is crucial, which is approximately 10 to 20 micrometers. Test the size on the double sided tape and then apply it to the injection site. Using this procedure, we can take advantage of the photo switchable property of DiD probe to obtain super resolution images of the plasma membrane.This way we can study the ultra structural characterization of the synaptic membranes at the neuromuscular junction. We did the click. We performed phenotypic analysis of the aCC dendrites in mutant strain, and discover that a Dscam1-Dock-Pak injection defines the site of the dendrite outgrowth in aCC.

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In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

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 ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...