I thank Alex L. Kolodkin, as I learned this filleted preparation protocol in his laboratory. I also thank Young Gi Hong for technical assistance. &#160;This study was supported by NRF-2013R1A1A4A01011329 (S.J.).

1. Preparation
<ol>
	<li>Prepare 500 mL of phosphate-buffered saline (PBS) with t-Octylphenoxypolyethoxyethanol (PBT) solution by adding 0.5 g of bovine serum albumin (BSA) and 0.5 mL of t-Octylphenoxypolyethoxyethanol (see the Table of Materials) to 500 mL of 1X PBS and stirring for at least 30 min. Store at 4 &#176;C. Use when relatively fresh and store the solution in a clean bottle.</li>
	<li>Make 10 mL of 4% paraformaldehyde by adding 2.5 mL of 16% stock paraformaldehyde solution and 1 mL of 10x PBS to 6.5 mL of deionized water. Store at 4 &#176;C and use within one week. 
	CAUTION: Avoid skin contact with paraformaldehyde solution because it is highly toxic.</li>
	<li>Make X-Gal substrate (10% w/v X-Gal in dimethyl sulfoxide (DMSO)) by dissolving 0.1 g of X-Gal substrate per 1 mL of DMSO. Store at -20 &#176;C in 100 &#181;L aliquots.</li>
	<li>Make X-Gal staining solution using 10 mM phosphate buffer (pH 7.2), 150 mM NaCl, 1 mM MgCl2, 3 mM K4[FeII(CN)6], 3 mM K3[FeIII(CN)6], and 0.3% t-Octylphenoxypolyethoxyethanol. Store at 4 &#176;C in the dark.</li>
	<li>Make 3% hydrogen peroxide solution by diluting 30% stock hydrogen peroxide 1:10 with deionized water.</li>
	<li>Make 3,3&#8242;-diaminobenzidine (DAB) solution by completely dissolving 1 tablet (10 mg) of DAB in 35 mL of fresh PBT solution. Filter through a 0.2-&#181;m syringe filter and store at -20 &#176;C in 910 &#181;L aliquots. 
	CAUTION: Dispose of all DAB waste in bleach to destroy the DAB.</li>
	<li>Make egg plates with apple juice. Add 35 g of agar and 1 L of deionized water to a 2 L flask. Add 33 g of sucrose, 2 g of tegosept, and 375 mL of apple juice to a 1- L flask. Melt them by autoclaving for 30 min. Allow both solutions to cool to around 60 &#176;C. Combine and mix these solutions well by stirring. Pour ~11 mL of combined solution per 60 mm culture dish.</li>
	<li>Make yeast paste by blending baker yeast into deionized water (1:0.8 ratio) and store at 4 &#176;C.</li>
</ol>
2. Embryo Collection (Day 1)
<ol>
	<li>Collect young female and male flies (younger than 5 days), and for 1-2 days, maintain them in a plastic beaker cage with an egg plate (60 mm x 15 mm) that contains a small amount of yeast paste in the center.</li>
	<li>For the optimal collection of late stage-16 embryos, set up a bottle cage with flies (&#62; 50) at around 5 PM and let them lay eggs at 25 &#176;C for 3 h.</li>
	<li>Transfer the flies to a fresh food bottle.</li>
	<li>Close the egg plate with the lid and incubate it upside down at 25 &#176;C in a humidified incubator (~70% humidity) overnight.</li>
</ol>
3. Preparation of Embryos for Immunostaining (Day 2)
<ol>
	<li>Add 1.8 mL of PBT solution to the egg plate around 10:20 AM and use a cotton swab to loosen the embryos from the plate while tilting it. 
	Note: By gently mashing the yeast paste using a cotton swab, dissolve it in case a large number of embryos are found on it. Begin collecting the embryos ~40 min before fixation (around 11:00 AM).</li>
	<li>Transfer the embryos from the egg plate to a 1.5-mL microtube using a 1-mL pipette tip.</li>
	<li>Allow the embryos to settle down and aspirate the PBT solution as much as possible. Rinse the embryos twice with 1 mL of PBT solution. Aspirate the PBT solution as much as possible.</li>
	<li>Fill the tube with 1 mL of 50% bleach (i.e., diluted in deionized water) and dechorionate the embryos by incubating them on a nutator for 3 min at room temperature (RT). 
	Note: This step does not kill the dechorionated embryos.</li>
	<li>Allow the dechorionated embryos to settle down, aspirate the 50% bleach as much as possible, and rinse the embryos 3 times with 1 mL of PBT solution.</li>
	<li>Add 0.5 mL of heptane and subsequently add 0.5 mL of 4% paraformaldehyde solution to the tube at RT at around 11:00 AM.</li>
	<li>Incubate the embryos on a nutator for 15 min at RT.</li>
	<li>Remove the 4% paraformaldehyde solution (the bottom layer).</li>
	<li>Add 0.5 mL of 100% methanol and devitellinize the embryos by shaking the tube vigorously for 30 s.</li>
	<li>Remove the heptane (i.e., the top layer).</li>
	<li>Add 0.5 mL of 100% methanol and shake the tube vigorously for 10 s.</li>
	<li>Allow the embryos to settle down by tapping the tube and aspirate the methanol as much as possible. Rinse the embryos with 1 mL of 100% methanol, shaking for less than 10 s. 
	Note: Extended exposure to methanol destroys &#946;-Gal activity.</li>
	<li>Remove the methanol and rinse the devitellinized embryos 3 times with 1 mL of PBT solution.</li>
</ol>
4. Genotyping the Embryos Using LacZ Staining (Day 2)
<ol>
	<li>Add 1 mL of PBT solution to the tube and incubate the tube in a 37 &#176;C heat block or water bath for 15 min.</li>
	<li>During incubation, place X-Gal staining solution (1 mL per tube) at 65 &#176;C in a water bath until it gets cloudy. Incubate it in a 37 &#176;C water bath.</li>
	<li>Aspirate the PBT solution and add 1 mL of X-Gal staining solution and 20 &#181;L of X-Gal substrate to the tube.</li>
	<li>Incubate the tube at 37 &#176;C with nutation until a blue precipitate is evident. 
	Note: The incubation time for the 2nd and 3rd blue balancers in general ranges from 2-4 h.</li>
	<li>Remove the X-Gal staining solution and rinse the embryos 3 times with 1 mL of RT PBT solution.</li>
	<li>Using a needle probe or forceps, hand-sort the embryos of the desired genotype (i.e., non-stained white embryos) with a light dissecting microscope (1.6X-2.5X objectives). 
	Note: Since some blue balancers, such as CyO, actin-LacZ and TM3, actin-LacZ, carry a transgenic construct expressing bacterial &#946;-Gal (LacZ) under the control of the actin promoter, embryos stained blue in a ubiquitous manner contain one or two copies of blue balancer chromosomes. Therefore, non-stained white embryos are homozygous for the desired lethal allele.</li>
</ol>
5. Immunostaining of Embryos with Anti-Fasciclin II Antibodies (Day 2-3)
<ol>
	<li>Collect and transfer the embryos of the desired genotype (i.e., non-stained white embryos) to a 0.5 mL microtube using a 1 mL pipette tip. 
	Note: In this step, the embryos can be roughly staged according to morphological criteria, including the segmentation patterns of the cuticle and the structures of the head and tail9. During head involution, between 10 and 16 h after the eggs are laid, the embryo undergoes a pronounced reduction of the most anterior segment9.</li>
	<li>Wash the embryos once with 0.4 mL of PBT solution and add 0.3 mL of blocking solution (5% normal goat serum and 5% DMSO in PBT solution).</li>
	<li>Block the embryos with nutation at RT for 15 min.</li>
	<li>Add 75 &#181;L of anti-Fasciclin II antibody and incubate the tube with nutation at RT overnight.</li>
	<li>Wash the embryos 4 times with 0.4 mL of PBT solution for at least 20 min per wash.</li>
	<li>Add 0.3 mL of blocking solution (5% normal goat serum in PBT solution) containing goat anti-mouse-HRP antibody (2 &#181;g/mL) and incubate the tube with nutation at RT overnight.</li>
	<li>Wash the embryos 6 times with 0.4 mL of PBT solution for at least 20 min per wash.</li>
	<li>Add 0.3 mL of DAB solution to the tube. 
	CAUTION: Wear gloves and place all DAB wastes/tubes/tips into bleach.</li>
	<li>Add 2 &#181;L of 3% hydrogen peroxide and incubate the tube in the dark with nutation at RT until the desired amount of precipitate is produced. 
	Note: The incubation time for 1D4 immunohistochemistry in general ranges from 0.5-1 h.</li>
	<li>Wash the embryos 4 times with 0.4 mL of PBT solution. 
	CAUTION: Remove the DAB solution and the first two washes with a pipette and discard them in bleach.</li>
	<li>Add 0.2 mL of mounting 70% glycerol/PBS solution to the tube and store at RT or 4 &#176;C. 
	Note: Keep the tube away from light. Clearer preparations can be obtained by placing the embryos in a 90% glycerol/PBS solution10. However, it is more difficult to dissect embryos cleared in 90% glycerol/PBS solution than those cleared in 70% glycerol/PBS solution10.</li>
</ol>
6. Staging, Dissecting, Mounting, and Imaging the Embryos (Day 4)
<ol>
	<li>For staging, transfer the embryos equilibrated with 70% glycerol/PBS onto a glass slide.</li>
	<li>Collect late stage-16 embryos that have abdominal segment 7 (A7), A8, and A9 ISN nerves converging to touch each other or/and have midgut that is subdivided into three bands. 
	Note: Embryos stained with 1D4 antibody can be staged based on the projection patterns of ISN and ISNb motor axons. After exiting the CNS, A7, A8, and A9 ISN nerves of late stage-16 embryos converge to touch each other and subsequently diverge to extend far out to the dorsal muscles (arrowhead in Figure 1A). In mid-stage-16 embryos, however, A7 ISN nerves extend in parallel with A8/A9 nerves (square bracket in Figure 1B). In addition, the projection axes of A6 ISNb nerves (perpendicular to the ventrolateral muscles) in both hemisegments are roughly aligned with the posterior end of the CNS longitudinal axon fascicles (arrows and a line in Figure 1C). These morphological criteria somewhat vary between different genotypes. Alternatively, embryos can be staged based on gut morphology9,11. Prior to stage 17, the midgut has a heart-like shape, whereas the midgut is subdivided into three bands by stage 1712.</li>
	<li>Dissect the embryos.
	<ol>
		<li>Using a 1 mL syringe, move each embryo out of the glycerol drop and place the embryo ventral-face up. Cut off the anterior part at 1/4 of the body length and the most posterior region that is free of motor axons.</li>
		<li>Using a needle probe, move the end-cut embryo out of the glycerol drop, roll to orient it dorsal-side up, and place it horizontally in the field. 
		Note: When the embryo is moved out of the glycerol drop, a little bit of glycerol associated with the embryo makes it sticky.</li>
		<li>Cut the embryo along the dorsal midline using a single very fine tungsten needle13. Make a small cut at the posterior end of the embryo and then continue to cut down the dorsal midline toward the anterior; cutting in the opposite direction is also fine.</li>
		<li>Using a needle probe, move the midline-cut embryo into the glycerol drop, place it dorsal-side up, and detach the gut from the body wall by unfolding each body wall in a ventrolateral (diagonal) direction (2.5X objective). 
		Note: Make sure that the dorsal midline is fully cut, resulting in the complete separation between the left and right body walls.</li>
		<li>Carefully move the midline-cut embryo out of the glycerol drop and then lay two flaps of the body wall down on the glass slide using a fine needle probe (2.5X objective).</li>
		<li>Using a very fine tungsten needle, remove the internal organs from the dissected embryo by pushing them laterally (5X objective). 
		Note: A more detailed description of another similar dissection procedure can be found on the website5.</li>
	</ol>
	</li>
	<li>For mounting, add 2-3 &#181;L of 70% glycerol/PBS solution to clean, dissected embryos and, using a fine needle probe, transfer them to a new glass slide onto which 8 &#181;L of 70% glycerol/PBS solution has been spread in the center (2.5X objective).</li>
	<li>Put on a coverslip (18 mm x 18 mm). Seal the edges of the coverslip with regular nail polish (either clear or colored is fine).</li>
	<li>Capture images at high resolution (20X, 40X, and 63X oil-immersion objectives) under a differential interference contrast (DIC) light microscope, according to the manufacturer&#39;s instructions. 
	Note: Better visualization and phenotypic characterization, especially for ISNb motor axons, can be produced by using a 63X oil-immersion objective.</li>
</ol>

<ol><li>Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+embryonic+development+of+larval+muscles+in+Drosophila%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The embryonic development of larval muscles in Drosophila.</a> Development. 110 (3), 791-804 (1990).</li>
<li>Landgraf, M., Thor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+Drosophila+motoneurons%3A+specification+and+morphology%5BTitle%5D)+AND+%22Semin.+Cell+Dev.+Biol%22%5BJournal%5D)">Development of Drosophila motoneurons: specification and morphology.</a> Semin. Cell Dev. Biol. 17 (1), 3-11 (2006).</li>
<li>Grenningloh, G., Rehm, E. J., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+analysis+of+growth+cone+guidance+in+Drosophila%3A+fasciclin+II+functions+as+a+neuronal+recognition+molecule%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Genetic analysis of growth cone guidance in Drosophila: fasciclin II functions as a neuronal recognition molecule.</a> Cell. 67 (1), 45-57 (1991).</li>
<li>Vactor, D. V., Sink, H., Fambrough, D., Tsoo, R., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genes+that+control+neuromuscular+specificity+in+Drosophila%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Genes that control neuromuscular specificity in Drosophila.</a> Cell. 73 (6), 1137-1153 (1993).</li>
<li>Zinn Lab. , California Institute of Technology. Available from:
	 <a target="_blank" href="https://www.its.caltech.edu/~zinnlab/motoraxons.html">https://www.its.caltech.edu/~zinnlab/motoraxons.html</a> (2017).</li>
<li>Seeger, M., Tear, G., Ferres-Marco, D., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutations+affecting+growth+cone+guidance+in+Drosophila%3A+genes+necessary+for+guidance+toward+or+away+from+the+midline%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline.</a> Neuron. 10 (3), 409-426 (1993).</li>
<li>Thor, S., Thomas, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Drosophila+islet+gene+governs+axon+pathfinding+and+neurotransmitter+identity%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The Drosophila islet gene governs axon pathfinding and neurotransmitter identity.</a> Neuron. 18 (3), 397-409 (1997).</li>
<li>Roh, S., Yang, D. S., Jeong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+ligand+regulation+of+PlexB+signaling+in+motor+neuron+axon+guidance+in+Drosophila%5BTitle%5D)+AND+%22Int.+J.+Dev.+Neurosci%22%5BJournal%5D)">Differential ligand regulation of PlexB signaling in motor neuron axon guidance in Drosophila.</a> Int. J. Dev. Neurosci. 55, 34-40 (2016).</li>
<li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCampos-Ortega%2C+JA+%22The+embryonic+development+of+Drosophila+melanogaster%22">The embryonic development of Drosophila melanogaster</a>. , Springer-Verlag. New York. (1985).</li>
<li>Patel, N. H. Imaging neuronal subsets and other cell types in whole mount Drosophila embryos and larvae using antibody probes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPatel%2C+NH+%22Methods+in+cell+biology%2C+vol+44.+Drosophila+melanogaster%3A+practical+uses+in+cell+biology%22">Methods in cell biology, vol 44. Drosophila melanogaster: practical uses in cell biology</a>. Goldstein, L. S. B., Fyrberg, E. 44, Academic Press. New York. 445-487 (1994).</li>
<li>Lee, H. K., Wright, A. P., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+dissection+of+Drosophila+embryos%3A+streamlined+methods+for+screening+mutant+collections+by+antibody+staining%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Live dissection of Drosophila embryos: streamlined methods for screening mutant collections by antibody staining.</a> J. Vis. Exp. (34), (2009).</li>
<li>Hartenstein, V. Stages of Embryonic Development. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHartenstein%2C+V+%22Atlas+of+Drosophila.+development%22">Atlas of Drosophila. development</a>. , Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. Available from:
 <a target="_blank" href="http://www.sdbonline.org/sites/fly/atlas/52.htm">http://www.sdbonline.org/sites/fly/atlas/52.htm</a> 52(1993).</li>
<li>Brady, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+technique+for+making+very+fine%2C+durable+dissecting+needles+by+sharpening+tungsten+wire+electrolytically%5BTitle%5D)+AND+%22Bull.+World+Health+Organ%22%5BJournal%5D)">A simple technique for making very fine, durable dissecting needles by sharpening tungsten wire electrolytically.</a> Bull. World Health Organ. 32 (1), 143-144 (1965).</li>
<li>Kolodkin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fasciclin+IV%3A+sequence%2C+expression%2C+and+function+during+growth+cone+guidance+in+the+grasshopper+embryo%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Fasciclin IV: sequence, expression, and function during growth cone guidance in the grasshopper embryo.</a> Neuron. 9 (5), 831-845 (1992).</li>
<li>Jeong, S., Juhaszova, K., Kolodkin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Control+of+semaphorin-1a-mediated+reverse+signaling+by+opposing+pebble+and+RhoGAPp190+functions+in+Drosophila%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The Control of semaphorin-1a-mediated reverse signaling by opposing pebble and RhoGAPp190 functions in Drosophila.</a> Neuron. 76 (4), 721-734 (2012).</li>
<li>Winberg, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plexin+A+is+a+neuronal+semaphorin+receptor+that+controls+axon+guidance%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Plexin A is a neuronal semaphorin receptor that controls axon guidance.</a> Cell. 95 (7), 903-916 (1998).</li>
<li>Yang, D. S., Roh, S., Jeong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+axon+guidance+function+of+Rap1+small+GTPase+is+independent+of+PlexA+RasGAP+activity+in+Drosophila%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">The axon guidance function of Rap1 small GTPase is independent of PlexA RasGAP activity in Drosophila.</a> Dev. Biol. 418 (2), 258-267 (2016).</li>
<li>Yu, H. H., Araj, H. H., Ralls, S. A., Kolodkin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+transmembrane+Semaphorin+Sema+I+is+required+in+Drosophila+for+embryonic+motor+and+CNS+axon+guidance%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The transmembrane Semaphorin Sema I is required in Drosophila for embryonic motor and CNS axon guidance.</a> Neuron. 20 (2), 207-220 (1998).</li>
<li>Hartenstein, V. Stages of Embryonic Development. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHartenstein%2C+V+%22Atlas+of+Drosophila.+development%22">Atlas of Drosophila. development</a>. , Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. Available from:
 <a target="_blank" href="http://www.sdbonline.org/sites/fly/atlas/52.htm">http://www.sdbonline.org/sites/fly/atlas/52.htm</a> 52(1993).</li>
<li>Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanisms+of+axon+guidance%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Molecular mechanisms of axon guidance.</a> Science. 298 (5600), 1959-1964 (2002).</li>
<li>Kidd, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roundabout+controls+axon+crossing+of+the+CNS+midline+and+defines+a+novel+subfamily+of+evolutionarily+conserved+guidance+receptors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors.</a> Cell. 92 (2), 205-215 (1998).</li>
<li>Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Getting+neural+circuits+into+shape+with+semaphorins%5BTitle%5D)+AND+%22Nat.+Rev.+Neurosci%22%5BJournal%5D)">Getting neural circuits into shape with semaphorins.</a> Nat. Rev. Neurosci. 13 (9), 605-618 (2012).</li>
</ol>

The author declares that he has no competing financial interests.

The details of motor axon guidance defects are scored faster and with better accuracy by the filleted preparation of DAB-stained embryos than by laser scanning confocal microscopy of fluorescently labeled ones. Therefore, the filleted preparation of fixed and 1D4-stained embryos is best suited for the functional characterization of guidance cue molecules. Four major classes of guidance cues, including netrins, Slits, semaphorins (Semas), and ephrins, and their cognate receptors have evolutionarily conserved roles across ...

Precise and selective connections between motor axons and target muscles during embryonic development are essential for normal locomotion in Drosophila larvae. The embryonic patterning of 30 muscle fibers in each of the abdominal hemisegments A2-A7 is established by stage 161. The 36 motor neurons that are generated in the ventral nerve cord extend their axons into the periphery to innervate specific target muscles2. Motor axon pathfinding and target recognition can be visualized by immunohistochemistry with an antibody (mouse monoclonal antibody 1D4)3,4. Multiple images of the motor axon projection patterns in wildtype embryos are available on the web5. The 1D4 antibody labels all motor axons and three longitudinal axon fascicles on each side of the midline of the embryonic central nervous system (CNS)4,6 (Figure 1C and Figure 2A). Therefore, immunohistochemistry with FasII antibody provides a powerful tool for identifying genes required for neuromuscular connectivity for demonstrating the molecular mechanisms underlying motor axon guidance and target recognition.
In each of the abdominal hemisegments A2-A7, motor axons project and selectively fasciculate into two principal nerve branches, the segmental nerve (SN) and the intersegmental nerve (ISN)2,4, and a minor nerve branch, the transverse nerve (TN)7. The SN selectively defasciculates to give rise to two nerve branches called the SNa and SNc, whereas the ISN splits into three nerve branches called the ISN, ISNb, and ISNd2,4. Among them, ISN, ISNb, and SNa motor axon projection patterns are most precisely visualized when late stage-16 embryos are stained with FasII antibody and are filleted (Figure 1C and Figure 2A). The ISN motor neurons extend their axons to innervate dorsal muscles 1, 2, 3, 4, 9, 10, 11, 18, 19, and 202,4 (Figure 2A). The ISNb motor neurons innervate ventrolateral muscles 6, 7, 12, 13, 14, 28, and 302,4 (Figure 2A and 2B). The SNa nerve branch projects to innervate lateral muscles 5, 8, 21, 22, 23, and 242,4 (Figure 2A). The TN, which consists of two motor axons, projects ipsilaterally along the segmental border to innervate muscle 25 and makes synapses with the lateral bipolar dendritic neuron (LBD) in the periphery7 (Figure 2A). These target muscle innervations require not only selective defasciculation of motor axons at specific choice points, but also target muscle recognition. In addition, some putative mesodermal guidepost cells that act as intermediate targets were found in both the ISN and SNa pathways, but not along the ISNb pathway4. This might suggest that ISNb motor axon pathfinding can be regulated in a distinct manner compared to ISN and SNa motor axon guidance, and it also indicates that peripheral motor axon guidance provides an attractive experimental model to study the differential or conserved roles of a single guidance cue molecule8.
This work presents a standard method to visualize the axonal projection patterns of embryonic motor neurons in Drosophila. The described protocols include how to dissect fixed embryos stained with 1D4 antibody and processed in 3,3&#8242;-diaminobenzidine (DAB) for filleted preparations. One critical advantage of the flat preparations of fixed embryos is the better visualization of the axonal projections and muscle patterns in the periphery. Furthermore, this work also shows how to genotype fixed embryos to sort the desired mutant embryos using the LacZ staining method.

<table><tbody><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>t-Octylphenoxypolyethoxyethanol</td></tr><tr><td>16% Paraformaldehyde Solution</td><td>Ted Pella</td><td>18505</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P5405</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma-Aldrich</td><td>30435</td><td></td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma-Aldrich</td><td>71500</td><td></td></tr><tr><td>X-Gal Substrate</td><td>US Biological</td><td>X1000</td><td>X-Gal (5-Bromo-4-chloro-3-indolyl-b-D-galactoside galactopyranoside)</td></tr><tr><td>Dimethyl Sulfxide</td><td>Sigma-Aldrich</td><td>D4540</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Potassium hexacyanoferrate(II) trihydrate</td><td>Sigma-Aldrich</td><td>P9387</td><td></td></tr><tr><td>Potassium hexacyanoferrate(III)</td><td>Sigma-Aldrich</td><td>244023</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Sigma-Aldrich</td><td>216763</td><td></td></tr><tr><td>3,3&#039;-diaminobenzidine Tetrahydrochloride</td><td>Sigma-Aldrich</td><td>D5905</td><td></td></tr><tr><td>Agar</td><td>US Biological</td><td>A0930</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-3</td><td></td></tr><tr><td>Tegosept (Methy 4-Hydroxybenzoate)</td><td>Sigma-Aldrich</td><td>H5501</td><td></td></tr><tr><td>Culture Dish (60 mm)</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>Tricon Beaker</td><td>Simport</td><td>B700-100</td><td>This is used to make a plastic beaker cage for embryo collection.</td></tr><tr><td>Yeast</td><td>Societe Industrielle Lesaffre</td><td>Saf Instant Yeast Red</td><td></td></tr><tr><td>Cotton Swab (Wooden Single Tip Cotton PK100)</td><td>VWR</td><td>14220-263</td><td></td></tr><tr><td>Eppendorf Tube (1.5 ml)</td><td>Sarstedt</td><td>#72.690</td><td></td></tr><tr><td>Bleach</td><td>The Clorox Company</td><td>Clorox</td><td></td></tr><tr><td>Heptane</td><td>Sigma-Aldrich</td><td>246654</td><td></td></tr><tr><td>Methanol</td><td>J.T. Baker</td><td>UN1230</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Life Technologies</td><td>16210-064</td><td></td></tr><tr><td>Anti-FasciculinII Antibody</td><td>Developmental Studies Hybridoma Bank</td><td>1D4 anti-Fasciclin II</td><td></td></tr><tr><td>Goat Anti-mouse-HRP Antibody</td><td>Jackson Immunoresearch</td><td>115-006-068</td><td>AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Goat Anti-Mouse IgG+IgM (H+L)&lt;br/&gt; (min X Hu, Bov, Hrs Sr Prot</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G9012</td><td></td></tr><tr><td>Slide Glass</td><td>Duran Group</td><td>235501403</td><td></td></tr><tr><td>Coverslip</td><td>Duran Group</td><td>235503104</td><td>18 x 18 mm</td></tr><tr><td>1 ml Syringe</td><td>Becton Dickinson Medical(s)</td><td>301321</td><td></td></tr><tr><td>Tungsten Needle</td><td>Ted Pella</td><td>#27-11</td><td>Tungsten Wire, &amp;oslash;0.13mm/6.1m (&amp;oslash;.005&quot;/20 ft.)</td></tr><tr><td>Nutator (Mini twister)</td><td>Korean Science</td><td>KO.VS-96TWS</td><td>Alternatively, BD Clay Adams Brand Nutator (BD 421125)</td></tr></tbody></table>

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.

Precise connections between motor axons and target muscles during neural development depend on selective axon-axon repulsion and target recognition at specific choice points4. In Drosophila, selective repulsion between motor axons is in part regulated by the combined action of class 1 and 2 semaphorins (Semas), including Sema-1a, Sema-2a, and Sema-2b8,14,15,<...

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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.

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 ...

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Research

JoVE Journal

Neuroscience

7.8K Views.  Chonbuk National University.  Dit werk bevat een standaard immunohistochemie methode om motorische neuron projecties van late-16 Drosophila melanogaster embryo's te visualiseren . De gefiltreerde bereiding van vaste embryo's die gekleurd zijn met FasII antilichaam, biedt een krachtig instrument om de genen die nodig zijn voor motor axon pathfinding en target recognition tijdens neurale ontwikkeling te karakteriseren. 

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

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

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

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

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

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

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

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 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 ...

Developmental Biology

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection 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 ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Retrograde Labeling of Drosophila Motor Neurons Using Fluorescent Lipophilic Dyes

<a href='https://app.jove.com/v/60716/retrograde-tracing-drosophila-embryonic-motor-neurons-using'>Source: Inal, M. A. et. al., Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes. J. Vis. Exp. (2020)</a>This video demonstrates the method of retrograde labeling of Drosophila motor neurons using lipophilic dyes. The dye enters the axon, travels retrogradely, and labels the neurons green, allowing detailed visualization of their morphology and projections under a confocal microscope.

Source: Inal, M. A. et. al., Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes. J. Vis. Exp. (2020)This video ...

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

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