We thank Karine Kindbeiter, Muriel Bozon, and Florie Reynaud for their help. The work is supported by CNRS, ANR YODA program and Labex DevWeCan.

DAY 1
1. Dissection of the Spinal Cord Floor Plate (FP) from E12.5 Mouse Embryos
Note: The entire procedure requires the use of sterile conditions. It is preferable to perform the dissection under a dissection hood to avoid contamination. The surface of the hood should be cleaned with ethanol. All dissection instruments must be sterilized and kept in a sterile Petri dish. The liquids (medium, dissection medium) must be kept closed&#160;and put in an ice bath. Each embryo should be collected in individual fresh drop. This is important for tissue preservation. The dissection is performed with Dumont #5 forceps. To transfer the embryos between dishes, grip the umbilical cord or the head without any damage to the hindbrain. It is critical not to damage the spinal cord to successfully complete the dissection. Prepare cold Phosphate Buffered Saline (PBS), cold Hank&#39;s Balanced Salt Solution (HBSS)-6.5% glucose and room temperature neurobasal medium.
1.1 Spinal cord dissection
Animals are treated according to the animal care guidelines of the Centre National de la Recherche Scientifique following European directives. The method of euthanasia is cervical dislocation, it consist of applying pressure to the neck and dislocating the spinal column from the brain. This method is recommended for mouse animal models, and can be performed in a fast and efficient way.
<ol>
	<li>Euthanize a pregnant mouse at E12.5 of gestation (E0.5= first day following matting). Place the mouse ventral side up.</li>
	<li>Soak the abdomen with 70% ethanol. Pinch the skin of the abdomen with Adson forceps and cut through the skin and the peritoneal layers with surgical scissors. Make an incision and use it to cut on both side of the mouse to expose the abdominal cavity.</li>
	<li>A string of embryos is present on each side of the mouse. Grasp one horn of the uterus in the tissue between two embryos and dissect it out starting from the exterior part. Transfer the intact uterus to a 100 mm Petri dish with cold PBS on ice.</li>
	<li>Extract the embryos from extra-embryonic tissues. Grasp the placenta tissue (in dark red) with two pairs of forceps and pull softly to tear the tissue and remove the embryos from the uterine sac.</li>
</ol>
Note: For a better conservation, collect the embryos from their uterine sac one by one.
<ol start="5">
	<li>The next steps are done under binocular magnifying glass and dissection hood. Place one embryo in a drop of cold HBSS-6.5% glucose.</li>
	<li>Using forceps, decapitate the embryo.</li>
</ol>
Note: Cut between the lower jaw and the hindbrain, the posterior part of the hindbrain is important for the next steps.
<ol start="7">
	<li>Place the embryo ventral face down with the anterior part facing the experimenter.</li>
	<li>Pinch the skin of the embryo with one pair of forceps and with the other pair pull away the skin.</li>
</ol>
Note: This step has to be repeated until the skin is completely removed from the anterior part of the embryo.
<ol start="9">
	<li>Turn the embryo anterior side to the left. With one forceps &#34;pin down&#34; the embryo in the anterior part. With the other forceps grab the skin and pull towards the rostral side of the embryo. The spinal cord is then accessible.</li>
	<li>Use one side of the forceps to cut the meninges that still wrap the spinal cord. The spinal cord is now open.</li>
	<li>Turn the embryo anterior side to the right.</li>
	<li>Detach the tissue from the spinal cord starting from the cervical side of the embryo. Slide the forceps under the spinal cord (the forceps should be held closed to avoid spinal cord damage). The forceps have to be visible under the spinal cord. Small rotatory movements under the spinal cord are sufficient to detach the surrounding tissue and the dorsal root ganglia (DRG).</li>
</ol>
Note: Leaving attached surrounding tissue add weight to the spinal cord, thus increasing the probability of breaking it during the next step.
<ol start="13">
	<li>Place the isolated spinal cord in a fresh drop of cold HBSS-6.5% glucose.</li>
	<li>Place the spinal cord in a flat position, meninges on the top and anterior side away from the experimenter.</li>
	<li>&#34;Pin down&#34; the spinal cord using the remaining hindbrain with one forceps, and with the second forceps grab the meninges. Peel off the meninges starting from the rostral side of the spinal cord.</li>
</ol>
Note: The movement should be slow and constant to avoid any breaking of the spinal cord. Vibratory movements can facilitate the detachment of the meninges.
1.2 FP dissection
<ol>
	<li>Pinch the hindbrain with one forceps.</li>
	<li>The FP is the central transparent part of the tissue. Using a scalpel cut out the floor plate.</li>
</ol>
Note: To cut both sides with the same quality, start from the rostral side and alternately cut the right side and the left side of the floor plate.
<ol start="3">
	<li>Conserve the dissected FP in Neurobasal medium at room temperature.</li>
</ol>
2. FP Culture
Note: All steps should be performed under sterile conditions in a tissue culture hood. Use fresh medium and freshly thawed supplements and reagents. Prepare sterile glass coverslips, warm neurobasal+B27, plasma, and HBSS-thrombin.
2.1 FP culture
<ol>
	<li>Place several sterile coverslips in a 100 mm Petri dish and pipette 20 &#956;l plasma in the center of each coverslip.</li>
	<li>Transfer two to four FP in each plasma drop and add 20 &#956;l HBSS-thrombin next to the plasma drop and carefully mix. Keep the coverslips for a minimum of ten minutes at room temperature to allow coagulation of the plasma.</li>
</ol>
Note: Transfer two FPs in case of complete dissection and more in case of partial dissection. Mix slowly with a pipette tip and avoid contact with the FP explants.
The plasma clot retracts a few &#956;m as a sign of the coagulation. The coagulation must not be stopped prematurely, to avoid detachment of the plasma clot.
<ol start="3">
	<li>Transfer each coverslip in a 24-well plate and add 500 &#956;l of Neurobasal+B27.&#160;Incubate 48 hr at 37 &#176;C.</li>
</ol>
DAY 3
3. FP Conditioned Medium (FPcm) Harvesting
Note: Harvesting should be performed under sterile conditions in a tissue culture hood. If the supernatant is contaminated or if floor plates are floating (meaning they might have died during the culture period) then do not harvest the FPcm from this well.
3.1 FPcm harvesting
<ol>
	<li>Harvest carefully the medium from each well.</li>
</ol>
Note: Do not pipette the polymerized plasma-thrombin mix or any FP fragments.
<ol start="2">
	<li>Store 100 &#956;l aliquots at -80 &#176;C.</li>
</ol>
DAY 4
4. Dissection of Spinal Cord Commissural Neurons from E12.5 Mouse Embryos
Note: This preparation does not need to be performed under sterile conditions. Follow the procedure described above to collect the embryos. Prepare cold PBS, cold HBSS-6.5% glucose and cold neurobasal.
4.1 Spinal cord dissection
<ol>
	<li>Using forceps, decapitate the embryo.</li>
</ol>
Note: Cut between the lower jaw and the hindbrain, the posterior part of the hindbrain is important for the next steps.
<ol start="2">
	<li>Place the embryo ventral face down in a fresh drop with the anterior part facing the experimenter.</li>
	<li>Pinch the skin of the embryo with one forceps and with the other one pull away the skin.</li>
</ol>
Note: This step has to be repeated until the skin is completely removed from the anterior part of the embryo.
<ol start="4">
	<li>Turn the embryo anterior side to the left. With one forceps &#34;pin down&#34; the embryo in the anterior part. With the other forceps, grasp the skin and pull towards the rostral side of the embryo, to expose the spinal cord.</li>
	<li>Use one side of the forceps to cut the meninges that still wrap the spinal cord. The spinal cord is now exposed and open.</li>
	<li>Position the embryo anterior side to the right.</li>
	<li>Detach the tissue from the spinal cord starting from the cervical side of the embryo. Slide the forceps under the spinal cord (keep the forceps closed). Forceps must visible under the spinal cord. Small rotatory movements under the spinal cord are sufficient to detach the adjacent tissue and the dorsal root ganglia (DRG).</li>
	<li>Transfer the isolated spinal cord in a new drop of cold HBSS-6.5% glucose.</li>
	<li>Place the spinal cord in a flat position, meninges on the top and anterior side away from the experimenter.</li>
	<li>&#34;Pin down&#34; the spinal cord using the remaining hindbrain with one forceps, with the second forceps grab the meninges. Peel off the meninges starting from the rostral side of the spinal cord.</li>
</ol>
4.2 Commissural Neuron dissection
<ol>
	<li>Pinch the hindbrain with one forceps.</li>
	<li>The Commissural neurons are located in the most lateral part of the spinal cord (the dorsal side). Cut out this lateral part with a fine scalpel.</li>
</ol>
Note: The dorsal part of the spinal cord can be distinguished from the ventral part by difference of cell density, the ventral part being more opaque.
Note: To cut both sides with the same quality, start from the rostral side and alternately cut a small length of the right side and of the left side.
<ol start="3">
	<li>Conserve the dissected tissue in Neurobasal medium on ice for immediate use.</li>
</ol>
5. Treatment of Commissural Tissue with the FPcm
Note: This procedure does not require sterile conditions. The tissue should be collected in individual 1.5 ml tubes. The frozen FPcm should be used only once. Avoid multiple refreezing. Prepare warm FPcm (37 &#176;C).
5.1 Treatments of commissural neurons
<ol>
	<li>Dilacerate the commissural tissue with small scissors.</li>
</ol>
Note: Cut the tissue as small as possible, to potentiate the accessibility of the cells.
<ol start="2">
	<li>Distribute fairly the tissue between the different experimental conditions control treatment and FPcm treatment.</li>
	<li>Centrifuge the tissue fragments at 800 x g for 1 min at 4 &#176;C.</li>
	<li>Check the repartition between tubes.</li>
</ol>
Note: If necessary redistribute and centrifuge again until fragments are equally distributed.
<ol start="5">
	<li>Remove the supernatant.</li>
	<li>Add the warm FPcm and control treatments to the tissues.</li>
</ol>
Note: The volume must be adjusted to the amount of the pellet and should be at least twice the volume of the pellet.
<ol start="7">
	<li>Incubate at 37 &#176;C for 30 min.</li>
</ol>
Note: Gently shake every 10-15 min
<ol start="8">
	<li>Centrifuge at 800 x g for 1 min and remove the treatment.</li>
	<li>Add the lysis buffer</li>
</ol>
Note: The volume must be adjusted to the amount of the pellet and should be at least twice the volume of the pellet.
<ol start="10">
	<li>Pipette up and down to resuspend the pellet.</li>
</ol>
Note: A vortex can also be used.
<ol start="11">
	<li>Incubate 15 min on ice.</li>
	<li>Centrifuge at 14,000 x g for 15 min at 4 &#176;C.</li>
	<li>Keep the supernatant and measure the amount of protein with a Bradford assay9.</li>
	<li>Add 6x&#160;Laemmli Buffer.</li>
	<li>Incubate at 95 &#176;C for 5 min to denature the protein content.</li>
</ol>
Note: Samples can be used immediately or can be stored at -80 &#176;C.
<ol start="16">
	<li>Assess the effects of the treatment by Western blot10.</li>
</ol>
Note: At least 50 &#956;g of total proteins should be loaded for each lane.

<ol>
	<li>Nawabi, H., Castellani, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+commissures+in+the+central+nervous+system:+how+to+cross+the+midline.">Axonal commissures in the central nervous system: how to cross the midline.</a> Cell. Mol. Life Sci. 68, 2539-2553 (2011).</li><li>Placzek, M., Briscoe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+floor+plate:+multiple+cells,+multiple+signals.">The floor plate: multiple cells, multiple signals.</a> Nat. Rev. Neurosci. 6, 230-240 (2005).</li><li>Chedotal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+tales+of+the+midline.">Further tales of the midline.</a> Curr. Opin. Neurobiol. 21, 68-75 (1016).</li><li>Nawabi, 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=A+midline+switch+of+receptor+processing+regulates+commissural+axon+guidance+in+vertebrates.">A midline switch of receptor processing regulates commissural axon guidance in vertebrates.</a> Genes Dev. 24, 396-410 (2010).</li><li>Ruiz de Almodovar, 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=VEGF+mediates+commissural+axon+chemoattraction+through+its+receptor+Flk1.">VEGF mediates commissural axon chemoattraction through its receptor Flk1.</a> Neuron. 70, 966-978 (2011).</li><li>Charoy, 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=gdnf+activates+midline+repulsion+by+Semaphorin3B+via+NCAM+during+commissural+axon+guidance.">gdnf activates midline repulsion by Semaphorin3B via NCAM during commissural axon guidance.</a> Neuron. 75, 1051-1066 (2012).</li><li>Kiecker, C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+organizers+in+patterning+the+nervous+system.">The role of organizers in patterning the nervous system.</a> Annu. Rev. Neurosci. 35, 347-367 (2012).</li><li>Le Dreau, G., Marti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+patterning+of+the+neural+tube:+a+tale+of+three+signals.">Dorsal-ventral patterning of the neural tube: a tale of three signals.</a> Dev. Neurobiol. 72, 1471-1481 (2012).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J. Vis. Exp. , (2010).</li><li>Salinas, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+collaborates+with+netrin-1+to+guide+axons+in+the+spinal+cord.">The morphogen sonic hedgehog collaborates with netrin-1 to guide axons in the spinal cord.</a> Trends Neurosci. 26, 641-643 (2003).</li><li>Bechara, 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=FAK-MAPK-dependent+adhesion+disassembly+downstream+of+L1+contributes+to+semaphorin3A-induced+collapse.">FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse.</a> EMBO J. 27, 1549-1562 (2008).</li></ol>

The authors have nothing to disclose

The production of floor-plate conditioned medium provides an efficient and easy way for assessing the biological properties of floor plate released signals.
The plasma-thrombin matrix provides excellent condition for tissue survival. Nevertheless, this enriched environment might be a limitation for some types of experiments. Thus, the floor plate tissue can also be cultivated in agarose matrix. The quality of the floor plate conditioned medium can be assessed by the detection of known floor-pl...

The floor-plate is a well-known patterning center of the developing spinal cord, playing key roles in the specification of progenitors and postmitotic cell lineages and controlling axon navigation7,8. The experimental procedure described here to produce FPcm allows investigators to assess the functional properties of floor plate-derived signals in a various contexts of developmental processes, from cell patterning and survival to cell and axon migration.
To illustrate the use of such FPcm, dorsal spinal cord tissues containing commissural neurons are dissected, dilacerated and stimulated with the FPcm. The tissues can then be processed for Western blot analysis. This allows investigating regulations of the axon guidance machinery by floor-plate released signals. The method of treatment of dilacerated fresh tissue holds the great advantage of preserving the microenvironment of cells within the tissue. Thus the consequences of the treatment by FPcm or any types of treatment are assessed in a more physiological way than in cell and tissue culture conditions.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS REQUIRED</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, Ca2+/Mg2+- free</td>
			<td>Invitrogen</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G-7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Invitrogen</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td>Sigma</td>
			<td>P-3266</td>
			<td>1mg/ml (Reconstitute in H2O and filtrate)</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T-4648</td>
			<td>10mg/ml(Reconstitute in H2O and filtrate)</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50X</td>
		</tr>
		<tr>
			<td>Hepes (260g/mol)</td>
			<td>Sigma</td>
			<td>H-7006</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E-5513</td>
			<td>0,5M pH8</td>
		</tr>
		<tr>
			<td>MgCL2</td>
			<td>Euromedex</td>
			<td>2189</td>
			<td>1M</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL</td>
			<td>Sigma</td>
			<td>I-3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Protease inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>04 693 116 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate (Na3VO4)</td>
			<td>Sigma</td>
			<td>S-6508</td>
			<td></td>
		</tr>
		<tr>
			<td>distilled H2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Reagents required</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>TOOLS AND MATERIAL</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Fine Tips forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>micro knives</td>
			<td>Fine Science Tools</td>
			<td>10136-14</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Tools and material</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			<table border="1">
				<tbody>
					<tr>
						<td colspan="2">Dissection and FP culture</td>
					</tr>
					<tr>
						<td>Ethanol 70%</td>
						<td></td>
					</tr>
					<tr>
						<td>dissection hood</td>
						<td></td>
					</tr>
					<tr>
						<td>dissection microscope</td>
						<td></td>
					</tr>
					<tr>
						<td>Petri dishes</td>
						<td></td>
					</tr>
					<tr>
						<td>glass coverslips</td>
						<td>18mm x 18mm</td>
					</tr>
					<tr>
						<td>Bunsen gas burner</td>
						<td></td>
					</tr>
					<tr>
						<td>24 well plate</td>
						<td></td>
					</tr>
					<tr>
						<td>tissue culture incubator (37&#176;C, 5% CO2, humidity controlled)</td>
						<td></td>
					</tr>
					<tr>
						<td>Centrifuge</td>
						<td></td>
					</tr>
					<tr>
						<td colspan="2">RECIPES</td>
					</tr>
					<tr>
						<td colspan="2">FP culture media</td>
					</tr>
					<tr>
						<td>Neurobasal</td>
						<td></td>
					</tr>
					<tr>
						<td>B27</td>
						<td>1/50e dilution from stock</td>
					</tr>
					<tr>
						<td colspan="2">HBSS - Thrombin</td>
					</tr>
					<tr>
						<td>HBSS</td>
						<td>1 ml</td>
					</tr>
					<tr>
						<td>Thrombin 10mg/ml</td>
						<td>30 &#956;l</td>
					</tr>
					<tr>
						<td colspan="2">Lysis Buffer pH7,4</td>
					</tr>
					<tr>
						<td>Hepes (260 g/mol)</td>
						<td>25mM</td>
					</tr>
					<tr>
						<td>EDTA (pH8)</td>
						<td>5mM</td>
					</tr>
					<tr>
						<td>MgCL2</td>
						<td>1mM</td>
					</tr>
					<tr>
						<td>Glycerol</td>
						<td>10%</td>
					</tr>
					<tr>
						<td>IGEPAL (NP40)</td>
						<td>0.10%</td>
					</tr>
					<tr>
						<td>Protease Inhibitor Cocktail</td>
						<td>1X</td>
					</tr>
					<tr>
						<td>Sodium Orthovanadate</td>
						<td>0,2M</td>
					</tr>
					<tr>
						<td>H2O</td>
						<td></td>
					</tr>
					<tr>
						<td colspan="2">Laemmli Buffer</td>
					</tr>
					<tr>
						<td>Tris 0,5M, pH 6,8</td>
						<td>375 mM</td>
					</tr>
					<tr>
						<td>SDS</td>
						<td>6%</td>
					</tr>
					<tr>
						<td>Glycerol</td>
						<td>60%</td>
					</tr>
					<tr>
						<td>DTT</td>
						<td>0,6M</td>
					</tr>
					<tr>
						<td>Bromophenol blue</td>
						<td></td>
					</tr>
					<tr>
						<td>H2O</td>
						<td></td>
					</tr>
				</tbody>
			</table>
			</td>
		</tr>
	</tbody>
</table>

culture of isolated floor plate tissue and production of conditioned medium to assess functional properties of floor plate-released signals

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of glial cells lining the ependymal canal at ventral position. The floor-plate expresses key morphogens contributing to the patterning of cell lineages in the spinal cord. At later developmental stages, the floor-plate regulates the navigation of axons in the spinal cord, acting as a barrier to prevent the crossing of ipsilateral axons and controlling midline crossing by commissural axons1. These functions are achieved through the secretion of various guidance cues. Some of these cues act as attractants and repellents for the growing axons while others regulate guidance receptors and downstream signaling to modulate the sensitivity of the axons to the local guidance cues2,3. Here we describe a method that allows investigating the properties of floor-plate derived signals in a variety of developmental contexts, based on the production of Floor-Plate conditioned medium (FPcm)4-6. We then exemplify the use of this FPcm in the context of axon guidance. First, the spinal cord is isolated from mouse embryo at E12.5 and the floor-plate is dissected out and cultivated in a plasma-thrombin matrix (Figure 1). Second two days later, commissural tissue are dissected out from E12.5 embryos, triturated and exposed to the FPcm. Third, the tissue are processed for Western blot analysis of commissural markers.

Commissural tissues were treated by the FPcm and the samples were processed for analysis of receptor levels in Western blot. Application of the FPcm was shown to increase the levels of a guidance receptor, PlexinA1, which mediates the sensitivity of commissural axons to the midline repellent Semaphorin3B after crossing (Figure 2). This revealed that signals released by the FP regulate PlexinA1 levels4,6.
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Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

The floor plate is a crucial structure of the developing spinal cord, providing diffusible signals to progenitors and differentiating neurons. We describe a method to produce floor-plate conditioned medium, to apply it to fresh spinal cord tissue, and to assess the consequences on proteins of interest by biochemistry.

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of ...

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Research

JoVE Journal

Neuroscience

7.9K Views.  University of Lyon 1. The floor plate is a crucial structure of the developing spinal cord, providing diffusible signals to progenitors and differentiating neurons. We describe a method to produce floor-plate conditioned medium, to apply it to fresh spinal cord tissue, and to assess the consequences on proteins of interest by biochemistry.