Name: visualization of the axonal projection pattern of embryonic motor neurons in drosophila
Uploaded: 2017-06-16T14:00:00
Description: Watch this Scientific Journal Video about Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila at JoVE.com

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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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 ...

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Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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

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 ...

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...