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 m...

<ol>
	<li>Davis-Dusenbery, B. N., Williams, L. A., Klim, J. R., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+spinal+motor+neurons.">How to make spinal motor neurons.</a> Development. 141, 491-501 (2014).</li><li>Stifani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neurons+and+the+generation+of+spinal+motor+neuron+diversity.">Motor neurons and the generation of spinal motor neuron diversity.</a> Front Cell Neurosci. 8, 293 (2014).</li><li>Kania, A., 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=Topographic+motor+projections+in+the+limb+imposed+by+LIM+homeodomain+protein+regulation+of+ephrin-A:EphA+interactions.">Topographic motor projections in the limb imposed by LIM homeodomain protein regulation of ephrin-A:EphA interactions.</a> Neuron. 38, 581-596 (2003).</li><li>Livet, 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=ETS+gene+Pea3+controls+the+central+position+and+terminal+arborization+of+specific+motor+neuron+pools.">ETS gene Pea3 controls the central position and terminal arborization of specific motor neuron pools.</a> Neuron. 35, 877-892 (2002).</li><li>Kanning, K. C., Kaplan, A., Henderson, C. 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. M., Lee, D. H., Liu, J. P., Dasen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+Proteins+Coordinate+Motor+Neuron+Differentiation+and+Connectivity+Programs+through+Ret/Gfralpha+Genes.">Hox Proteins Coordinate Motor Neuron Differentiation and Connectivity Programs through Ret/Gfralpha Genes.</a> Cell Rep. 14, 1901-1915 (2016).</li><li>Bonanomi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ret+is+a+multifunctional+coreceptor+that+integrates+diffusible-+and+contact-axon+guidance+signals.">Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals.</a> Cell. 148, 568-582 (2012).</li><li>Tung, Y. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Any+Way+You+Slice+It-A+Comparison+of+Confocal+Microscopy+Techniques.">Any Way You Slice It-A Comparison of Confocal Microscopy Techniques.</a> J Biomol Tech. 26, 54-65 (2015).</li><li>Reynaud, E. G., Peychl, J., Huisken, J., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+to+light-sheet+microscopy+for+adventurous+biologists.">Guide to light-sheet microscopy for adventurous biologists.</a> Nat Methods. 12, 30-34 (2015).</li><li>Santi, P. 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. G., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meeting+report:+first+light+sheet+based+fluorescence+microscopy+workshop.">Meeting report: first light sheet based fluorescence microscopy workshop.</a> Biotechnol J. 5, 798-804 (2010).</li></ol>

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 ...

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 axo...

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 ...

The overall goal of this protocol is to allow qualitative and quantitative assessment of motor neuron axon arborization patterns using light sheet microscope and image analysis software on mouse embryos. This method can help to understand motor neuron development in coordination with complex movement. The main advantage of this technique is that you can observe and image axon arborization patterns of motor neurons from various angles and quantitatively analyze each individual nerve.Demonstrating the procedure will be Ya-Ping Yen and Ee Shan Liau, two graduate students from my laboratory. To begin this procedure, fix the embryos individually in a 24-well plate with one milliliter of freshly prepared 4%paraformaldehyde in PBS per well at four degrees Celsius on a shaker overnight. The next day, wash the fixed embryos at least three times, each for five to 10 minutes, with one milliliter of 1X PBS and incubate them overnight at four degrees Celsius.Next, permeabilize each embryo in one milliliter of 0.5%PBST overnight at four degrees Celsius on a shaker. Block each sample with one milliliter of 10%FBS prepared in 1X PBS overnight at 4 degrees Celsius on a shaker. Subsequently, incubate each embryo with one milliliter of anti-GFP primary antibody at 4 degrees Celsius for 72 hours with constant shaking.After the primary antibody incubation, wash each embryo with one milliliter of 0.5%PBST over the course of one to two days at four degrees Celsius on a shaker. Afterward, apply one milliliter of secondary antibody and incubate overnight in the dark at four degrees Celsius with constant shaking. The following day, wash each embryo with one milliliter of 0.5%PBST at least three times at four degrees Celsius on a shaker over the course of two to three days.Then, incubate each embryo in a commercial clearing reagent with a refractive index of 1.49 nd in a 1.5 milliliter tube protected from light at room temperature overnight. Set up 5X/0.1 illumination optics and 5X/0.16 detection optics. Assemble the sample holder and sample capillary.Prepare the sample chamber by filling it with clearing solution. To mount the embryo, prepare a P200 pipette tip by cutting away the upper part so that it fits the diameter of the capillary and remove the pointed part for the sample attachment. Then, melt the blunted end of the pipette tip using a small flame.Extinguish the flame and quickly attach the embryo vertically onto the melted end. Fit the upper part of the pipette tip with the sample capillary and put the prepared sample chamber into the microscope. Using the imaging software, select Locate Capillary under the Locate tab and adjust the X, Y, and Z axes.Next, select Locate Sample and zoom to 0.6X to focus on the embryo. Allow the chamber buffer to equilibrate with the embryo for some time to clear off any debris or bubbles. For image acquisition under the Acquisition tab, define the lightpath parameters such as Detective Objectives, Laser Blocking Filter, Beam Splitter, Cameras, and Lasers.Check the Pivot Scan check box for shadow reduction. Then, define the acquisition settings, Bit Bepth to 16-bit, Zoom to the range between 0.36 and 0.7X, single site illumination or dual site illumination. Click Continuous and set the Laser Intensity, Exposure Time, Laser Power, and Light Sheet Position.Subsequently, press Stop to end image acquisition. It is important to ensure that the transparent sample is positioned within the shortest lightpath to allow detailed acquisition of images of fine arborized structures on individual motor nerves. For multi-dimensional acquisition, define the Z-stack by moving the z position for the first and last image.Click Optimal to set the slice number. Then, click Start Experiment to acquire the selected Z-stack. When it is done, save the image in czi format.To quantify axon arborization, open the image file in the imagining analysis software. Adjust the image color, brightness, and contrast using the Display Adjustment window to detect filaments based on local intensity contrast. Next, click on the Add New Filaments icon and select the Autopath Algorithm in the dropdown menu.Select the region of interest, then define the starting and seed points by assigning the largest and thinnest diameter measurements which can be measured using the Slice mode. 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 and right-click at the points of interest.Next, select manual thresholds for seed points to ensure that all of the visible arborization is marked. Then, manually add or remove seed points. Check the box Remove Disconnected Segments and remove seed points around starting points.Subsequently, adjust the threshold for background subtraction and finish the process. It is necessary to remove background artifacts and confirm the detector path reflect true axon arborization for accurate quantification. At the end, choose the desired style and color.Under the Statistics tab, select Detailed and use filament number, dendrite terminal points to quantify the motor nerve terminals as an indicator of motor axon arborization. Then, export the image of the reconstructed axon as a tiff file. LSFM provides detailed 3D visualization of motor axon arborization in mouse embryos.Motor neurons are subjected to the above-described protocol and labeled with transgenically-expressed GFP. To image axon arborization in greater detail and to perform quantification, magnification can be adjusted so that every finely arborized structure can be revealed. Finally, axon arborization pattern of individual forelimb nerves can be reconstructed using image analysis software.The autopath algorithm in the software traces filament and quantifies the total number of axon terminals. After watching this video, you should have a good understanding of how to prepare samples and image motor neuron axons of mouse embryos using light sheet fluorescense microscope. Also, you will know how to quantitatively assess the arborization pattern of each individual motor nerves by MRS.

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7.6K Views.  National Defense Medical Center. The overall goal of this protocol is to allow qualitative and quantitative assessment of motor neuron axon arborization patterns using light sheet microscope and image analysis software on mouse embryos. This method can help to understand motor neuron development in coordination with complex movement. The main advantage of this technique is that you can observe and image axon arborization patterns of motor neurons from various angles and quantitatively analyze each individual nerve.Dem...