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 d...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transmembrane+Semaphorin+Sema+I+is+required+in+Drosophila+for+embryonic+motor+and+CNS+axon+guidance.">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+Embryonic+Development.">Stages of Embryonic Development.</a> Atlas of Drosophila. development. , 52 (1993).</li><li>Dickson, B. 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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.

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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,<...

Watch this Scientific Journal Video about Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila at JoVE.com

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 ...

The overall goal of this experiment is to analyze the motor neuron projection patterns in late stage embryos of Drosophila melanogaster. This method can help answer key questions in the field of neural development, such as motor axon guidance and neural subcu formation. The main advantage of this technique is that it provides precise visualization of motor axon in developing embryos, which allows for reliable analysis of axonal pathfinding and target the condition defect.One day after setting-up egg collection plates replace the plates with new ones containing a dab of freshly-made yeast paste. After three hours on the fresh plates, transfer the flies to new food bottles. And incubate the egg plates overnight at 25 degrees Celsius.In the morning, add 1.8 milliliters of PBT solution to the plates. And use a cotton swab to dislodge the embryos. Then, using a 1 milliliter pipette, transfer the embryos and solution to a micro centrifuge tube.Once the embryos settle to the bottom, aspirate as much solution as possible. Then rinse the embryos twice using one milliliter of PBT per rinse. To decoreonate the embryos, replace the last rinse with one milliliter of 50%bleach and rock the tube for three minutes at room temperature.To remove the bleach, rinse the embryos three times with PBT. Then, replace the last rinse with a half milliliter of 100%heptane and a half milliliter of 4%paraformaldehyde. Now, incubate the embryos for 15 minutes with gentle rocking.Following the incubation, remove the underlying solution layer and add back 500 microliters of 100%methanol. Now, for 30 seconds, shake the tube vigorously to devitellinize the embryos. Then, remove the overlying solution, add back 500 microliters of 100%methanol and shake the tube for another 10 seconds.After the shaking the tube, tap it to help the embryos settle and remove as much solution as possible. Next, briefly rinse the embryos with 100%methanol. Then, immediately rinse the embryos with PBT three times.Now, resuspend the embryos in one milliliter of fresh PBT. And incubate the embryos at 37 degrees Celsius for 15 minutes to prepare them for LacZ staining. While the embryos incubate, prepare a one milliliter aliquot of X-gal staining solution.Warm the aliquot in a 65 degree Celsius water bath until it gets cloudy. Then transfer it to a 37 degree Celsius heat block. After the embryos have been warmed for 15 minutes remove the PBT solution, and replace it with the one milliliter aliquot of staining solution.Then add 20 microliters of X-gal substrate. For about two to four hours, incubate the tube at 37 degrees Celsius with gentle rocking until a blue precipitate forms. Then, remove the staining solution and rinse the embryos three times with room temperature PBT.After the rinses, use a needle probe or forceps to hand-sort the embryos under a dissection microscope. Collect the stained embryos of interest into a half millimeter micro centrifuge tube using a one milliliter pipette. Next, wash the selected embryos with 0.4 milliliters of PBT.Then, replace the PBT with 0.3 milliliters of blocking solution and allow the embryos to incubate with gentle agitation for 15 minutes. After 15 minutes, add 75 microliters of the primary antibody and continue the incubation overnight. The following morning, wash the embryos with PBT for about two hours, changing the wash solution approximately every 30 minutes.After the wash, add 0.3 milliliters of blocking solution containing the secondary antibody and incubate the tube with gentle rocking at room temperature overnight. The next morning, wash the embryos with PBT for about three hours. Change the PBT at least five times during the wash period.After the wash, resuspend the embryos in 0.3 milliliters of dab solution and add two microliters of 3%hydrogen peroxide. Then, incubate them in the dark with gentle rocking at room temperature until enough precipitate is produced. This generally takes 30 to 60 minutes.Then, rinse the embryos with PBT four times and resuspend them in 0.2 milliliters of mounting solution. Now, allow the embryos to equilibrate in the dark at 4 degrees Celsius. To filet the embryos, stage them on a glass slide.First move them into a drop of glycerol ventral side up. Next, under a dissection microscope, cut off the anterior part and 1/4 of the body length and remove the most posterior region, which is free of motor axons. Now, using a needle probe, move the preparation out of the glycerol, oriented dorsal side up and position it horizontally in the field of view.The next step requires a fine tungsten needle that has been inserted into the hollow region of a needle probe, and then bent at the end. Using the tungsten needle, make a small cut at the posterior end of the embryo and continue to cut down the dorsal midline. Make sure the tip of the tungsten needle does not touch the surface of the glass slide during the dissection, because the needle will easily bend against the glass.Then, with the probe return preparation back to the glycerol drop and position it dorsal side up. There, detach the gut from the body wall by unfolding each body wall in a ventral-lateral direction. The two body walls should come apart.Now using the probe, carefully move the body wall flaps onto a glass slide. On the slide, use a tungsten needle to remove the internal organs by pushing them laterally. To stabilize the tungsten needle and keep it from being damaged, keep the hollow needle probed and hold the tungsten needle braced against the surface of the glass slide while manipulating the tungsten needle.Now, mount the preparations in eight microliters of mounting solution on a new glass slide. Attach a cover slip and seal the edges down with regular nail polish. Then, capture images at high resolution using DIC optics.Using the described method, stage 16 embryos were stained with Fas2 antibody and prepared for visualization of the motor neurons and abdominal hemi-segments A3 and A4.Normally, at least seven motor neurons extend and fasciculate their axons to form an ISNb nerve branch. Many surrounding nerve bundles were also identifiable in this preparation. When the intersegmental nerve bundle reaches the choice point at muscle 6 and 7, two axons selectively de-fasciculate and enervate muscles 6 and 7.The same occurs at other choice points as the bundle extends dorsally. Then, at the last choice point, two axons enervate muscle 12. By contrast, in Sema-1a mutant embryos the same axons fail to defasciculate at their choice points.This is not unexpected as the Sema-1 aegean product is known to help form connections between motor axons and target muscles during neural development. Thus, the described method can be used to analyze mutant phenotypes. After watching this video, you should have a good understanding of how to sort the desired mutant embryos, how to immunostain the axonal projection patterns of motor neurons, and how to dissect fixed embryos for flat preparation.

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Research

JoVE Journal

Neuroscience

7.6K vistas.  Chonbuk National University. The overall goal of this experiment is to analyze the motor neuron projection patterns in late stage embryos of Drosophila melanogaster. This method can help answer key questions in the field of neural development, such as motor axon guidance and neural subcu formation. The main advantage of this technique is that it provides precise visualization of motor axon in developing embryos, which allows for reliable analysis of axonal pathfinding and target the condition defect.One day after ...