Funding provided by the National Eye Institute [5K08EY027850], National Institute of Child Health and Development [U54HD090255], Harvard-Vision Clinical Scientist Development Program [5K12EY016335], the Knights Templar Eye Foundation [Career Starter Grant], and the Children&#8217;s Hospital Ophthalmology Foundation [Faculty Discovery Award]. ECE is a Howard Hughes Medical Institute investigator.

All animal work described here was approved and performed in compliance with the Boston Children&#39;s Hospital Institutional Animal Care and Use Committee (IACUC) protocols.
1. Timed matings
<ol>
	<li>Place ISLMN:GFP (Islet Motor Neuron Green Fluorescent Protein; MGI: J:132726; Jax Tg(Isl-EGFP*)1Slp/J Stock No:&#160;017952) male and female mice together overnight. Weigh the females and record weights prior to mating. 
	NOTE: ISL...

<ol>
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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=Neuropilin-2+is+required+in+vivo+for+selective+axon+guidance+responses+to+secreted+semaphorins.">Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins.</a> Neuron. 25 (1), 29-41 (2000).</li><li>Chen, H., 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=Neuropilin-2+regulates+the+development+of+selective+cranial+and+sensory+nerves+and+hippocampal+mossy+fiber+projections.">Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections.</a> Neuron. 25 (1), 43-56 (2000).</li><li>Lerner, 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=Stromal+cell-derived+factor-1+and+hepatocyte+growth+factor+guide+axon+projections+to+the+extraocular+muscles.">Stromal cell-derived factor-1 and hepatocyte growth factor guide axon projections to the extraocular muscles.</a> Developmental Neurobiology. 70 (8), 549-564 (2010).</li><li>Cheng, L., 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=Human+CFEOM1+mutations+attenuate+KIF21A+autoinhibition+and+cause+oculomotor+axon+stalling.">Human CFEOM1 mutations attenuate KIF21A autoinhibition and cause oculomotor axon stalling.</a> Neuron. 82 (2), 334-349 (2014).</li><li>Tischfield, M. A., 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=Human+TUBB3+mutations+perturb+microtubule+dynamics,+kinesin+interactions,+and+axon+guidance.">Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance.</a> Cell. 140 (1), 74-87 (2010).</li><li>Kim, M., 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=Motor+neuron+cell+bodies+are+actively+positioned+by+Slit/Robo+repulsion+and+Netrin/DCC+attraction.">Motor neuron cell bodies are actively positioned by Slit/Robo repulsion and Netrin/DCC attraction.</a> Developmental Biology. 399 (1), 68-79 (2015).</li><li>Montague, K., Guthrie, S., Poparic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+and+In+Vitro+Knockdown+Approaches+in+the+Avian+Embryo+as+a+Means+to+Study+Semaphorin+Signaling.">In Vivo and In Vitro Knockdown Approaches in the Avian Embryo as a Means to Study Semaphorin Signaling.</a> Methods in molecular biology. 1493, 403-416 (2017).</li><li>Clark, C., Austen, O., Poparic, I., Guthrie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha2-Chimaerin+regulates+a+key+axon+guidance+transition+during+development+of+the+oculomotor+projection.">alpha2-Chimaerin regulates a key axon guidance transition during development of the oculomotor projection.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33 (42), 16540-16551 (2013).</li><li>Ferrario, J. E., 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=Axon+guidance+in+the+developing+ocular+motor+system+and+Duane+retraction+syndrome+depends+on+Semaphorin+signaling+via+alpha2-chimaerin.">Axon guidance in the developing ocular motor system and Duane retraction syndrome depends on Semaphorin signaling via alpha2-chimaerin.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (36), 14669-14674 (2012).</li><li>Dupin, I., Dahan, M., Studer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+axonal+guidance+with+microdevice-based+approaches.">Investigating axonal guidance with microdevice-based approaches.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33 (45), 17647-17655 (2013).</li><li>Ebendal, T., Jacobson, 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=In+vitro+assay+to+test+differential+substrate+affinities+of+growing+axons+and+migratory+cells.">In vitro assay to test differential substrate affinities of growing axons and migratory cells.</a> Experimental Brain Research. 81 (2), 283-287 (1990).</li><li>Joanne Wang, C., 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=A+microfluidics-based+turning+assay+reveals+complex+growth+cone+responses+to+integrated+gradients+of+substrate-bound+ECM+molecules+and+diffusible+guidance+cues.">A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues.</a> Lab Chip. 8 (2), 227-237 (2008).</li><li>Wittig, J. H., Ryan, A. F., Asbeck, P. 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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=Tangential+migration+in+neocortical+development.">Tangential migration in neocortical development.</a> Developmental Biology. 244 (1), 155-169 (2002).</li><li>Miquelajauregui, A., 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=LIM-homeobox+gene+Lhx5+is+required+for+normal+development+of+Cajal-Retzius+cells.">LIM-homeobox gene Lhx5 is required for normal development of Cajal-Retzius cells.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 30 (31), 10551-10562 (2010).</li><li>Garcia-Pena, C. 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The authors have nothing to disclose.

This ex vivo slice culture protocol provides significant advantages over traditional axon guidance assays23. The size of each cranial motor nucleus is not a limiting factor, and no difficult dissection is necessary. The endogenous microenvironment through which the axons travel is maintained, allowing modification of one signaling pathway while maintaining other signaling pathways. Additionally, effects can be assessed at different points along the axon trajectory. Since axon guidance requires mul...

The ocular motor system provides an elegant system for investigating axon guidance mechanisms. It is relatively uncomplicated, consisting of three cranial nerves innervating six extraocular muscles (EOMs) which move the eye, and the levator palpebrae superioris (LPS) which lifts the eyelid. The oculomotor nerve innervates the LPS and four EOMs - the inferior oblique and the medial, inferior, and superior rectus muscles. The other two nerves, the trochlear and abducens, each only innervate one muscle, the superior oblique and lateral rectus muscle, respectively. Eye movements provide an easy readout, showing if innervation was appropriate, missing, or aberrant. Additionally, there are human eye movement disorders that result from deficits in neuronal development or axon guidance, collectively termed the congenital cranial disinnervation disorders (CCDDs)1.
Despite these advantages, the ocular motor system is rarely used in axon guidance studies2,3,4,5,6,7,8,9,10, due to technical drawbacks. In vitro axon guidance assays have many disadvantages11. Co-culture assays, in which neuronal explants are cultured together with explants of target tissue12 or transfected cells13, depend on both symmetry of the explant and precise positioning between the explant and target tissue. Stripe assays14,15, in which two cues are laid down in alternating stripes and axons are assessed for preferential growth on one stripe, only indicate that one substrate is preferable to the other, not that either is attractive or repulsive, or physiologically relevant. Microfluidics chambers can form precise chemical gradients, but subject growing axons to shear stress16,17,18, which can affect their growth. Moreover, in each of these approaches, collecting explants or dissociated cells requires that outgrowing axons be axotomized and thus these assays actually examine axon regeneration, rather than initial axon outgrowth. Finally, these in vitro approaches remove the microenvironment that influences axons and their responses to cues along different points of their course, and traditionally only test one cue in isolation. Compounding these disadvantages, the small size of each nucleus in the ocular motor system makes dissection technically challenging for either explants or dissociated cultures. Additionally, primary cultures of ocular motor neurons are usually heterogeneous, have significant cell death, and are density dependent, requiring pooling of cells from multiple embryos (Ryosuki Fujiki, personal communication). In vivo methods, however, including knockout mouse models, are inappropriate to use for screening, given the time and expense required.
Methods developed to culture whole embryos19 allow labeling of migrating cells20 or blockade of specific molecules21, but whole embryo cultures require incubation in roller bottles which precludes real-time imaging of labeled structures. Surgical techniques that allow manipulation of the embryo and then subsequent further development either in the uterus or in the abdomen of the mother (maintaining the placental connection)22 are also possible, but these also do not allow time-lapse imaging.
To overcome the obstacles of in vitro assays and allow rapid screening of signaling pathways, an ex vivo embryonic slice culture technique was developed23, adapted from a previously published protocol for peripheral nerve outgrowth24. Using this protocol, the developing oculomotor nerve can be imaged over time in the presence of many of the surrounding structures along its trajectory, including EOM targets. By adding small molecule inhibitors, growth factors, or guidance cues to the culture media, we can assess guidance perturbations at multiple points along the axon trajectory, allowing more rapid assessment of potential growth and guidance factors.

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		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">24-Well Tissue Culture Plate</td>
			<td style="width:163px;">Genesee Scientific</td>
			<td style="width:344px;">25-107</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">6-Well Tissue Culture Plate</td>
			<td>Genesee Scientific</td>
			<td>25-105</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disposable Pasteur Pipet (Flint Glass)</td>
			<td>VWR</td>
			<td>14672-200</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11412-11</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluorobrite DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1896701</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glucose (200 g/L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2494001</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#39;s Balanced Salt Solution (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14175-095</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat Inactivated Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550H</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES Buffer Solution (1M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630106</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Glutamine (250 nM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Loctite Superglue</td>
			<td>Loctite</td>
			<td></td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Low Melting Point Agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>16520050</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Millicell Cell Culture Insert (30mm, hydrophilic PTFE, 0.4 um)</td>
			<td>Millipore Sigma</td>
			<td>PICM03050</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Moria Mini Perforated Spoon</td>
			<td>Fine Science Tools</td>
			<td>10370-19</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122&#160;</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Petri Dish (100 x 15mm)</td>
			<td>Genesee Scientific</td>
			<td>32-107G</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phosphate Buffered Saline (1X, pH 7.4)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Razor Blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical Scissors - Blunt</td>
			<td>Fine Science Tools</td>
			<td>14000-12</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ti Eclipse Perfect Focus with TIRF</td>
			<td>Nikon</td>
			<td></td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome (VT 1200S)</td>
			<td>Leica</td>
			<td>1491200S001</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome Blades (Double Edge, Stainless Steel)</td>
			<td>Ted Pella, Inc.</td>
			<td>121-6</td>
			<td style="width:331px;"></td>
		</tr>
	</tbody>
</table>

ex vivo oculomotor slice culture from embryonic gfp-expressing mice for time-lapse imaging of oculomotor nerve outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

Normal Results: Figure 1 provides a schematic of the experiment. Starting as early as E9.5 in mouse, the first axons begin to emerge from the oculomotor nucleus26. By E10.5, a fasciculated oculomotor nerve, which contains the early pioneer neurons, can be seen in the mesenchyme. There is significant variability between embryos at E10.5 (even within the same litter) in how far the nerve has progressed towards the orbit, likely due to developmental differences of a few ...

Watch this Scientific Journal Video about Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth at JoVE.com

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

This protocol allows us to identify axon guidance pathways active in the oculomotor nerve and to assess their roles at different points along the nerve trajectory in real time. This technique preserves the local environments through which axons travel and their final targets. The growing axons are not cut, so initial axon outgrowth, rather than regeneration, can be assessed.This method provides insights into axon guidance in the ocular motor system but could be adapted to the study of axon guidance of other nerves. This technique takes practice to master, and working quickly is extremely important. When trying it for the first time, use just a few embryos, rather than a whole litter.Before beginning the procedure, first confirm the pregnancy by ultrasound on embryonic day 10.5 from a timed mating. Harvest the embryos, spray the abdomen of the pregnant mouse with 70%ethanol, and use scissors to open the abdominal cavity to extract the uterus. Wash the uterus in a Petri dish of ice-cold HBSS before placing the organ in a second Petri dish of fresh ice-cold HBSS.Under a dissecting scope, remove the embryos from the uterine horn and their individual amniotic sacs, placing each embryo on the underside of the lid of a 12-well plate, on ice, as they are harvested. Use filter paper to remove any liquid surrounding each embryo, without touching the embryos themselves, and submerge the embryos in liquid 4%low-melting agarose. Place the plate lid on ice to solidify the agarose.When the agarose has hardened, flip over the embryos and cover the other side of each sample with additional agarose. When the second volume of agarose has solidified, use a fluorescence dissecting microscope to trim the agarose around each embryo so that each embryo will be oriented properly on the vibratome stage. The oculomotor nuclei and early axon outgrowths should be fluorescent.Align each embryo so that the nucleus, outgrowing axons, and eye form a line, and use a razor blade to trim the agarose parallel to this line. Next, fill the vibratome chamber with ice-cold slice buffer, and superglue the first embryo to the vibratome stage so that the blade will be parallel with the oculomotor nucleus and eyes. When the superglue is dry, submerge the vibratome stage so that the embryo is oriented facing away from blade, and use a new vibratome blade to obtain 400-to 450-micrometer slices.Use a sterile transfer pipette to transfer each slice into cold slicing buffer as it is acquired, and use the fluorescence dissecting microscope to select the slice containing the oculomotor nuclei and eyes. Using a sterile transfer pipette, transfer the slice onto a cell culture insert in a six-well plate containing 1.5 milliliters of culture medium per well, and place the plate in a 37-degree Celsius incubator. Remove the residual agarose from the vibratome stage, and superglue the next embryo onto the stage, continuing to collect slices until all of the embryos have been sectioned and plated.Then add the appropriate concentration of the inhibitor or recombinant molecule of interest, diluted in an appropriate solvent, to the medium of each well. Create a dose-response curve. Image the slices every 30 minutes by phase contrast and fluorescence microscopy for up to 72 hours.During normal in utero development, the first green fluorescent protein-positive oculomotor axons reach the orbit by embryonic day 11.5 before branching to their final targets, as observed in this representative slice culture. The orientation of the slice on the vibratome stage is crucial, as the slices are not interpretable if they're not oriented properly. For example, if the embryo is tilted to its side, only one oculomotor nucleus will be observed within the slice.If the embedded embryo is tilted too far towards its back, the eyes will not be present within the slice, and instead the upper limb and hindbrain or spinal cord may be included. It is also important to take care that during the placement of the slice onto the culture membrane, that the tissue does not become folded. Take care when dissolving the inhibitors and growth factors in organic solvents.For example, in this experiment, the slice died after the addition of ethanol to the medium. Remember to keep everything on ice and to minimize the time between the extraction of the embryos and placing the slices in the incubator. Using this technique, we have begun to identify additional axon guidance mechanisms at work in the ocular motor system.Remember to use caution when working with razor blades and that some inhibitors may be hazardous and should be handled carefully according to their safety profiles.

ex-vivo-oculomotor-slice-culture-from-embryonic-gfp-expressing-mice

Research

JoVE Journal

Neuroscience

8.2K vues.  Boston Children's Hospital. Ce protocole nous permet d’identifier les voies de guidage axon actives dans le nerf oculomotrice et d’évaluer leurs rôles à différents points le long de la trajectoire nerveuse en temps réel. Cette technique préserve les environnements locaux par lesquels les axones voyagent et leurs cibles finales. Les axones en croissance ne sont pas coupés, de sorte que la croissance initiale de l’axon, plutôt que la régénération, peut être évaluée.Cette méthode fournit un aper...