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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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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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. 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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 ...

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 12.5 (E12.5)</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 &amp;deg;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 1.49 clearing reagent</td><td>SunJin Lab</td><td>RC149001</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 ...

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

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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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Developmental Biology

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Biology

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

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 ...

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.

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.

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

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

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In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

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

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices