LSFM experiments and data analysis were performed in part using the advanced optical microscopes of the Division of Instrument Service in Academia Sinica and with the assistance of Ms. Shu-Chen Shen. We thank Ms. Sue-Ping Lee from the Imaging Core Facility of IMB for considerable technical assistance with Imaris image analysis. The IMB&#39;s Scientific English Editing Core reviewed the manuscript. This work is funded by Academia Sinica Career Development Award (CDA-107-L05), MoST (104-2311-B-001-030-MY3), and NHRI (NHRI-EX106-10315NC).

All of the live animals were kept in a specific pathogen free (SPF) animal facility, approved and overseen by IACUC of Academia Sinica.
1. Fixation
<ol>
	<li>Collect embryos of embryonic day 12.5 (E12.5) then decapitate and eviscerate them.</li>
	<li>Fix the embryos individually in 24-well plates with 1 mL/well of freshly prepared 4% paraformaldehyde in 1x phosphate buffer saline (PBS) overnight. Incubate at 4 &#176;C on a shaker.</li>
	<li>Wash the fixed embryos at least 3x, each for 5-10 min, with 1 mL of 1x PBS and incubate overnight at 4 &#176;C on a shaker to remove the residual paraformaldehyde.</li>
</ol>
2. Whole-mount Immunostaining
<ol>
	<li>Permeabilize each embryo in 1 mL of 0.5% PBS-Triton-X-100&#160;(PBST) overnight at 4 &#176;C on a shaker.</li>
	<li>Block each sample with 1 mL of 10% FBS prepared in 1x PBS overnight at 4 &#176;C on a shaker.</li>
	<li>Incubate the embryo with 1 mL of anti-GFP primary antibody (1:1,000 dilution in 10% FBS) at 4 &#176;C for 72 h with constant shaking to intensify the GFP signal of motor nerves.</li>
	<li>After the primary antibody incubation, wash with 1 mL of 0.5% PBST over the course of 1-2 days at 4 &#176;C on a shaker, changing the 0.5% PBST at least three times.</li>
	<li>Apply 1 mL of secondary antibody (1:1,000 dilution in 10% FBS) and incubate overnight in the dark at 4 &#176;C with constant shaking.</li>
	<li>Wash 3x with 1 mL of 0.5% PBST at 4 &#176;C on a shaker over the course of 2-3 days. Keep samples in 1x PBS at 4 &#176;C until the day before imaging. 
	NOTE: Embryos can be dissected into smaller segments before clearing, depending on which part of the embryos will be imaged. Forelimbs are preserved in this experimental approach (Figure 1A).</li>
	<li>Incubate the embryo in a commercial clearing reagent with a refractive index of 1.49&#160;nD in a 1.5 mL tube, protected from light at room temperature overnight, to render the embryo transparent for imaging. 
	NOTE: The volume of the clearing reagent is approximately five times that of the sample volume.</li>
</ol>
3. LSFM (2&#8211;3 h)
<ol>
	<li>Sample set-up
	<ol>
		<li>Set up&#160;5X/0.1 illumination optics and 5X/0.16 detection optics. Assemble the sample holder and sample capillary (1.5 mm internal diameter, green) and place them into the microscope. 
		NOTE: Refer to the microscope&#39;s Operating Manual for detailed set-up procedures.</li>
		<li>Mount the embryo as follows:
		<ol>
			<li>Prepare the P200 pipette tip by cutting away the upper part so that it fits the diameter of the capillary and remove the pointed part (~ 3 mm) for the sample attachment.</li>
			<li>Melt the blunted end of the pipette tip using a small flame.</li>
			<li>Extinguish the flame and quickly&#160;attach the embryo vertically onto the melted&#160;end.</li>
			<li>Fit the upper part of the pipette tip with the sample capillary.</li>
		</ol>
		</li>
		<li>Allow the chamber buffer to equilibrate with the embryo for 1 min to clear off debris or bubbles.</li>
		<li>Using the imaging software, select Locate capillary under the Locate tab and adjust the x, y, z axes to position the sample capillary.</li>
		<li>Select Locate sample and zoom to 0.6X to focus on the embryo. Rotate the embryo to ensure that the long axis of the imaged limb aligns with the light path of the two light sources (Figure 1B).</li>
	</ol>
	</li>
	<li>Image acquisition
	<ol>
		<li>Under the Acquisition tab, define the light path parameters such as detection objectives, laser blocking filter, beam splitter, cameras, and lasers. 
		NOTE: The GFP channel is used in this experiment (Excitation wavelength: 488 nm; emission filter: BP 505 to 545 nm, beam splitter: SPS LP 560).</li>
		<li>Check the pivot scan checkbox for shadow reduction.</li>
		<li>Define the acquisition settings: bit depth, 16 bit; zoom, 0.36-0.7X; single-side illumination (left/right) or dual-side illumination.</li>
		<li>Click Continuous and set the laser intensity, exposure time, laser power, and light sheet position to acquire sharper images. 
		NOTE: The laser power is kept as low as possible to minimize photodamage.</li>
		<li>Press STOP to end image acquisition.</li>
	</ol>
	</li>
	<li>Multidimensional acquisition
	<ol>
		<li>Define the z-stack by moving the Z position for the first and last image. Click Optimal to set the slice number.</li>
		<li>Click Start Experiment to acquire the selected z-stack.</li>
		<li>When it is done, save the image in .czi format.</li>
	</ol>
	</li>
	<li>Image processing (optional)
	<ol>
		<li>Proceed with Dual side fusion under the Lightsheet Processing channel if dual-side illumination is applied. Multiview processing is necessary if multi-views are acquired to combine images from multiple angles.</li>
		<li>Create a 2D image using data from the highest intensity pixels along the projection axis under the Maximum Intensity Projection channel.</li>
		<li>Under Copy channel, click Subset to select subsets of images from the original set.</li>
	</ol>
	</li>
</ol>
4. Quantification of Axon Arborization (30 Min for Each Individual Nerve)
<ol>
	<li>Open the image file in the imaging analysis software (by default, the image containing the XYZ data is opened in Surpass mode and as a 3D-rendered view).</li>
	<li>Adjust the image color, brightness, and contrast using the Display Adjustment window (Edit | Show Display Adjustment) to detect filaments based on local intensity contrast.</li>
	<li>Click on the Add New Filaments icon and select the Autopath (no loop) algorithm in the drop-down menu.</li>
	<li>Select the region of interest (in this case, the segmenting axon of interest). Click Next when finished.</li>
	<li>Define the starting and seed points by assigning the largest and thinnest diameter measurements, which can be measured using the Slice mode.</li>
	<li>Assign a starting point at the edge of the region of interest. To achieve manual addition or removal of starting points, first change the pointer mode (Navigate | Select) and then Shift + right-click at the points of interest.</li>
	<li>Select manual thresholds for seed points to ensure that all of the visible arborization is marked. Change the pointer mode (Navigate | Select) and then Shift + left-click at the points of interest to manually add or remove seed points. 
	NOTE: It is important to manually remove background noises and signals from neighboring nerves.</li>
	<li>Check the box Remove seed points around starting points.</li>
	<li>Check Remove disconnected segments to allow exclusion of points that are too far away and that may represent background noise. Indicate the Maximum Gap Length in the next step to define the upper limit for exclusion.</li>
	<li>Adjust the threshold for background subtraction, which uses a Gaussian filter to estimate the background intensity of every voxel.</li>
	<li>Set automatic threshold for dendrite diameter using the Approximate cross-section area algorithm.</li>
	<li>Skip the steps for spine calculation.</li>
	<li>Finish the process and choose the desired style&#160;and color.</li>
	<li>Uncheck the volume box in Properties to view only the reconstructed axons.</li>
	<li>Under the Statistics tab, select Detailed. Use Filament No. Dendrite Terminal Points to quantify the motor nerve terminals as an indicator of motor axon arborization. 
	NOTE: Statistical annotation can be added if desired.</li>
	<li>Export the image of the reconstructed axon as a .tif file.</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neuron+diversity+in+development+and+disease.">Motor neuron diversity in development and disease.</a> Annu Rev Neurosci. 33, 409-440 (2010).</li><li>Matsumoto, S., Kiryu-Seo, S., Kiyama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+Nerve+Arborization+Requires+Proteolytic+Domain+of+Damage-Induced+Neuronal+Endopeptidase+(DINE)+during+Development.">Motor Nerve Arborization Requires Proteolytic Domain of Damage-Induced Neuronal Endopeptidase (DINE) during Development.</a> J Neurosci. 36, 4744-4757 (2016).</li><li>Nagata, K., 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=ECEL1+mutation+implicates+impaired+axonal+arborization+of+motor+nerves+in+the+pathogenesis+of+distal+arthrogryposis.">ECEL1 mutation implicates impaired axonal arborization of motor nerves in the pathogenesis of distal arthrogryposis.</a> Acta Neuropathol. 132, 111-126 (2016).</li><li>Catela, C., Shin, M. 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T., 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=Mir-17+approximately+92+Governs+Motor+Neuron+Subtype+Survival+by+Mediating+Nuclear+PTEN.">Mir-17 approximately 92 Governs Motor Neuron Subtype Survival by Mediating Nuclear PTEN.</a> Cell Rep. 11, 1305-1318 (2015).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Jonkman, J., Brown, C. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+fluorescence+microscopy:+a+review.">Light sheet fluorescence microscopy: a review.</a> J Histochem Cytochem. 59, 129-138 (2011).</li><li>Hanley, O., 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=Parallel+Pbx-Dependent+Pathways+Govern+the+Coalescence+and+Fate+of+Motor+Columns.">Parallel Pbx-Dependent Pathways Govern the Coalescence and Fate of Motor Columns.</a> Neuron. 91, 1005-1020 (2016).</li><li>De Marco Garcia, N. V., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+motor+neuron+pool+identity+and+muscle+nerve+trajectory+defined+by+postmitotic+restrictions+in+Nkx6.1+activity.">Early motor neuron pool identity and muscle nerve trajectory defined by postmitotic restrictions in Nkx6.1 activity.</a> Neuron. 57, 217-231 (2008).</li><li>Li, C. J., 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=MicroRNA+filters+Hox+temporal+transcription+noise+to+confer+boundary+formation+in+the+spinal+cord.">MicroRNA filters Hox temporal transcription noise to confer boundary formation in the spinal cord.</a> Nat Commun. 8, 14685 (2017).</li><li>Swoger, J., Pampaloni, F., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-sheet-based+fluorescence+microscopy+for+three-dimensional+imaging+of+biological+samples.">Light-sheet-based fluorescence microscopy for three-dimensional imaging of biological samples.</a> Cold Spring Harb Protoc. 2014, 1-8 (2014).</li><li>Pantazis, P., Supatto, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+whole-embryo+imaging:+a+quantitative+transition+is+underway.">Advances in whole-embryo imaging: a quantitative transition is underway.</a> Nat Rev Mol Cell Biol. 15, 327-339 (2014).</li><li>Reynaud, E. 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The authors have nothing to disclose.

Several steps in the protocol may be subjected to changes under certain circumstances. For example, the duration of fixation depends on the age of the embryos, varying from 2 h to 1 or more days using freshly-made paraformaldehyde. Since fixation is carried out prior to whole mount immunostaining, for antibodies that are sensitive to protein crosslinking, methanol can be used as an alternative fixative agent. For a high signal-to-noise ratio upon staining, it is necessary to optimize the detergent concentration (between 0.1-0.5%). Additional washing steps before and after antibody incubation can also increase the signal-to-noise ratio. In addition, a tissue-clearing step is important to ensure that the light path is unblocked throughout deep axonal projections. To enhance tissue transparency, it is important to ensure minimal introduction of PBS during incubation in the commercial clearing agent, as the clearing effect is reversed by re-immersing samples in buffer solutions. Otherwise, a clearing reagent with a higher refractive index or a longer clearing period is recommended. Meanwhile, parameters during image acquisition vary among different samples. Dual-side illumination and multiview imaging may allow imaging with greater depth-of-field and enhanced detail. However, since illumination from different angles is acquired simultaneously, single-side illumination is more efficient for smaller specimens or moving samples. A good quality image ensures accuracy for the following quantitative analyses.
Previous studies, including those by our group, have utilized either confocal imaging or two-photon microscopy to observe the motor axon navigation process or arborization pattern in Hb9::GFP embryos10,15,16,17. Based on our experience, we believe LSFM to be superior to these other two methods since: 1) Imaging time is largely reduced as LSFM offers more rapid and higher quality imaging, whilst minimizing photodamage and phototoxicity of samples. Specimens are illuminated from the side with a thin light sheet, thereby creating an intrinsic optical section14,18,19. The center of the light sheet converges with the focal plane of the detection system, which is orthogonal to the illumination axis. Consequently, out-of-focus light is reduced and only fluorophores within the focal plane are illuminated for every image, i.e., rather than the entire specimen, thereby reducing photobleaching20. 2) Details of the arborization pattern are much better illustrated as LSFM allows multiview imaging. Instead of the specimen being mounted onto a slide, it is attached in such a way that rotation around a vertical axis is possible, as are movements along the x, y, and z dimensions. The images from the different planes and views are stacked to produce more isotropic 3D images. However, LSFM also has a limitation compared to confocal and two-photon approaches in terms of the massive file size created for each image. These pros and cons need to be weighed when deciding which imaging method to use, but ultimately the choice will depend on which microscopic hardware/software is available upon request.
In short, this protocol combines whole-mount immunostaining, state-of-the-art light sheet 3D imaging, together with quantification in image analysis software. We believe this protocol not only provides a new paradigm for visualizing motor axon navigation and trajectories in embryos with quantitation of axon arborization complexity but could also be applied to other neuron systems (i.e., sensory neurons) with appropriate transgenic mice.

Spinal MNs are the part of the central nervous system but innervate peripheral muscles to control movement. In the developing spinal cord, MN progenitors (pMNs) are established according to the signals emanating from the notochord and adjacent somites. All differentiated post-mitotic MNs are then generated from pMNs, eventually giving rise to a series of MN subtypes along the rostrocaudal axis of the spinal cord1,2. Spinal MNs are topographically and anatomically well organized. Their morphological arrangement correlates with the position of their respective target in the periphery3. Upon reaching their muscle targets, axons receive cellular and exogenous neurotrophic factors that induce them to extend and branch further into muscles. Innervation and branching defects may contribute to the failure to form neuromuscular junctions (NMJ). For example, glial-derived neurotrophic factors (GDNF)-induced Pea3 is indispensable for axon arborization into cutaneous maximus (CM) and latissimus dorsi (LD) muscles4,5. In addition, DINE knockout mouse embryos show defective arborization of phrenic nerves in the diaphragm, causing respiratory failure and mortality immediately after birth6,7. Therefore, this last step of MN maturation (i.e., axonal projection and branching) is critical to ensure communication between neurons and target cells.
To view arborization patterns, researchers normally conduct confocal imaging or two-photon fluorescence light microscopy of sectioned or whole-mount samples8,9,10. Both of these microscopy techniques generate acceptable resolution and depth penetration. Two-photon fluorescence light microscopy involves excitation of fluorophores by simultaneous absorption of two lower-energy photons11. Since two-photon excitation uses near-infrared radiation, the decreased excitation frequency contributes to reduced scattering and better tissue penetration up to 1 mm in tissue, thus allowing imaging with greater depth. Confocal microscopy removes by filters the out-of-focus signals and only collects light within the focal plane12. With this approach, images of samples from different focal planes can be combined to produce a three-dimensional (3D) image via a Z-stack function. Nevertheless, signal intensity is reduced as most of the light is blocked and high numerical apertures obscure the depth-of-field. More importantly, both techniques contribute to the severe photodamage and phototoxicity since the whole specimen receives illumination even when only one plane is imaged at a given time.
To circumvent these shortcomings, LSFM has become a favored alternative, with the advantages of being fast, light-efficient, and less phototoxic13,14. In addition, LSFM allows multi-view imaging. This approach is particularly suited to visualizing motor axons and their dispersing terminals as they spread through 3D space. LSFM outperforms the other two options because samples are mounted on a stage that allows rotation around a vertical axis and movements along the x, y, and z axes. This set-up not only allows for a minimally blocked view of the sample but also the choice of a desirable illumination path, a shortcoming of two-photon and confocal microscopy, both of which require mounting of samples on a flat slide. Therefore, LSFM is the most suitable tool for 3D imaging of axon arborization and for quantification of motor nerve terminals in mouse embryos.

<table>
	<tbody>
		<tr>
			<td>Hb9::GFP</td>
			<td>The Jackson labortory</td>
			<td>005029</td>
			<td>Collect embryos of embryonic day 13.5 (E13.5)</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde (PFA)</td>
			<td></td>
			<td></td>
			<td>For 200ml: Add 20ml 10X PBS, 8g PFA in ddH2O. Adjust pH to 7.4 with NaOH (10N). Filter sterilize and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline 10X (PBS 10X)</td>
			<td></td>
			<td></td>
			<td>For 1L: Add 80 g NaCl, 2 g KCl,14.4g Na2HPO4, 2.4 g KH2PO4 and top up with ddH2O. Autoclave and store at RT.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep polyclonal anti-GFP</td>
			<td>AbD Serotec</td>
			<td>4745-1051</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-sheep</td>
			<td>Invitrogen</td>
			<td>A-11015</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>RapiClear 1.47 clearing reagent</td>
			<td>SunJin Lab</td>
			<td>RC147001</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml micro tube</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>24 wells plate</td>
			<td>ThermoFisher</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>5 SA Tweezer</td>
			<td>ideal-tek</td>
			<td>3480641</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors striaght sharp/sharp</td>
			<td>Aesculap</td>
			<td>BC110R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgery Scalpels, single use</td>
			<td>Aesculap</td>
			<td>BA365</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>TKS</td>
			<td>RS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lightsheet Z.1 microscope</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 8.4.0 image analysis software</td>
			<td>Bitplane, Zurich, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B6.Cg-Tg(Hlxb9-GFP)1Tmj/J (Hb9::GFP mice)</td>
			<td>The Jackson Laboratory</td>
			<td>005029</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

LSFM provides detailed 3D visualization of MN trajectory and axon arborization in mouse embryos. Under bright field, tissues appear completely transparent after being immersed in the commercial clearing reagent. No shrinkage or swelling of the sample was noticed after up to one week of storage in clearing reagent before imaging. Under&#160;fluorescence channel, motor neurons are labeled with transgenically expressed GFP&#160;(Movie 1). In some instance, details of the axon structure are compromised. For example, Figure 2 represents low quality imaging. Several factors can impede imaging quality. Firstly, insufficient permeabilization and washing steps can lead to high background signal. Secondly, light scattering through inadequately cleared tissue will result in blurred images. Lastly, suboptimal sample positioning during image acquisition might increase light scattering or block the light path.
To image axon arborization in greater detail and to perform quantification, magnification can be adjusted so that every finely arborized structure can be revealed (Movie 2). Images of single- and dual-side illumination should be compared to determine the optimal resolution of arborization pattern (Figure 3). Imaging analysis software can then be used to reconstruct axon arborization patterns for individual forelimb nerves (Figure 4). By defining the starting point and the diameter for seed point detection, these arborized structures can be semi-automatically detected and computed while background signals are manually removed. We used the &#34;Filament - No. Dendrite Terminal Pts&#34; function mode to calculate the total motor nerve terminals of individual nerves as an indicator of motor axon arborization. Additionally, branch length, mean diameter, and volume can be calculated using &#34;Filament - Dendrite Length&#34;, &#34;Filament - Dendrite Mean Diameter&#34; and &#34;Filament - Dendrite Volume&#34;, respectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57546/57546fig1.jpg" /> 
Figure 1: Graphical illustration of sample set-up. (A) During sample preparation, embryos are first decapitated and eviscerated. Forelimbs are dissected along the dashed line. (B) In the sample chamber, samples are positioned to keep the whole region of interest along the light path. <a href="/files/ftp_upload/57546/57546fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/57546/57546vid1.jpg" /> 
Movie 1: Light sheet fluorescence microscopy of a large field of view of the upper half of an E12.5 transgenic mouse embryo (Hb9::GFP). The movie clearly demonstrates motor nerves projecting from the spinal cord to their innervating targets. The 3D image is first acquired under 0.3X magnification with single-side illumination and 30 ms exposure time and later is processed into video using image analysis software. Scale bar represents 500 &#181;m. <a href="/files/ftp_upload/57546/Movie1_whole_bra_view.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57546/57546fig2.jpg" /> 
Figure 2: Example of a low-quality image with high background signal and blurred regions (blue arrows). The improper steps during sample preparation and image acquisition may lead to the impaired image quality to jeopardize the accuracy of any subsequent quantification of axon arborization. Scale bar in the image represents 200 &#181;m. <a href="/files/ftp_upload/57546/57546fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 2" src="/files/ftp_upload/57546/57546vid2.jpg" /> 
Movie 2: Visualization of E12.5 forelimb nerves under 488 nm excitation with dual-side illumination at a magnification of 0.6X. The zoomed-in movie allows clear and panoramic visualization of every fine arborized structures on individual nerves. Scale bar represents 300 &#181;m. <a href="/files/ftp_upload/57546/Movie2_e125brwt1_.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57546/57546fig3v2.jpg" /> 
Figure 3: Comparison between single- and dual-side illumination. Both left and right illuminations display details of certain regions only (red arrows), whereas a combined image from dual-side illuminations provides a more complete view of the arborization pattern. However, single-side illumination can be achieved in a shorter imaging time and sometimes with better image quality. This is due to the fact that illumination paths from both sides may not lie perfectly on a single plane, resulting in blurring during dual-side illuminations. Images are shown as maximum intensity projection. Scale bar represents 200 &#181;m. <a href="/files/ftp_upload/57546/57546fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57546/57546fig4v2.jpg" /> 
Figure 4: Reconstructed axon arborization of individual forelimb nerves, color-coded as follows: suprascapular (red), axillary (pink), radial (blue), posterior brachial cutaneous (purple). Arborization complexity was calculated according to the number of terminal end-points detected using our semi-automated method. The Autopath (no loop) algorithm traces filaments and detects seed points from user-defined starting points. Scale bar in both images represents 200 &#181;m. <a href="/files/ftp_upload/57546/57546fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy at JoVE.com

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

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 ...

visualization-motor-axon-navigation-quantification-axon-arborization

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.6K Views.  National Defense Medical Center. 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.

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

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...