Name: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors (Video) | JoVE Journal
Uploaded: 2013-09-18T15:15:00.000+00:00
Duration: 8 min 26 s
Description: Learn a quantitative cell migration assay for murine enteric neural progenitors using fluorescence microscopy and collagen gel incubation. This method enables researchers to study growth factor effects on neural crest cell migration.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We thank Denis Flipo for image processing and analysis advice, and David W. Silversides in whose laboratory the Gata4p[5kb]-GFP mouse line was generated. Research in the Pilon laboratory is funded by CIHR, NSERC, FRQS and FRQNT.

Ethics statement
Experiments involving mice were performed following Canadian Council of Animal Care guidelines for the care and manipulation of animals used in medical research. Protocols involving the manipulation of animals were approved by the institutional ethics committee of the University of Quebec in Montreal (Comit&#233; Institutionnel de Protection des Animaux; reference number 0512-R3-650-0513).
1. Preparation of Collagen Gels
Work in a sterile fashion, under a tissue culture hood.
<ol>
	<li>Prepare complete 5x DMEM (Eagle's modified essential medium) including standard antibiotics. Dissolve 3.37 g of DMEM powder and 0.925 g of NaHCO3 in 20 ml water. Sterilize by passing through a 0.22 &mu;m filter. Add 2.5 ml of sterile 100x penicillin/streptomycin and 25 ml of sterile heat-inactivated fetal bovine serum. Store at 4 &#176;C.</li>
	<li>On ice, mix 800 &#181;l of Collagen I solution (3.77 mg/ml in 0.02 N acetic acid, filter-sterilized), 600 &#181;l of complete 5x DMEM and 17 &#181;l of 1 N NaOH. Dilute with sterile water to a final volume of 3 ml. Include relevant growth factors in the mix. We typically use GDNF at 10 ng/ml to stimulate enteric NCC migration.</li>
	<li>Deposit about 480 &#181;l in each well of a single row in a 24-well plate. Avoid bubbles. The remaining rows can be used to test the effect of other growth factors on cell migration.</li>
	<li>Let the collagen polymerize at least 1 hr in a sterile incubator at 37 &#176;C.</li>
</ol>
2. Dissection of Animals16
<ol>
	<li>Set up matings and check for vaginal plugs the next morning. Day e0.5 being noon on the day the vaginal plug is found, isolate the female and wait 12 days until e12.5.</li>
	<li>Anesthetize pregnant mice with isoflurane and euthanize by CO2 inhalation.</li>
	<li>Spray the mouse with 70% ethanol. Lift the abdomen skin and open the abdomen cavity with dissecting scissors.</li>
	<li>Remove the uterus into a glass Petri dish filled with ice-cold PBS (phosphate-buffered saline). Cut the uterus transversely between individual deciduum swellings to isolate each embryo implantation site.</li>
	<li>Work on each implantation site separately in another glass Petri dish filled with ice-cold PBS. Under a dissecting microscope, use fine forceps to remove the muscle layer of the uterus.</li>
	<li>Open the visceral yolk sac and amnion to reveal the embryo. Take care when severing the blood vessels joining the embryo to the placenta/visceral yolk sac, as they are intertwined with the developing intestines.</li>
	<li>Sever the embryo&#39;s head at the neck.</li>
	<li>Insert a closed forceps in the abdominal cavity of an embryo, just above the dark red-colored liver and let the forceps open of itself (stop applying pressure) to make a transversal opening in the abdomen cavity. Pull the opened forceps down towards the posterior end of the embryo to open the abdomen completely.</li>
	<li>Grab the connective tissue behind the liver and pull the guts out of the abdomen, being careful not to break the intestines (the colon is attached to the anus).</li>
	<li>Cut the colon to free the guts from the rest of the embryo. The cut can be made at any location along the colon. Reserve the tail portion for later.</li>
	<li>Tease out the connective tissue from the cecum, then the rest of the intestines. Be careful not to wound the intestines while doing so.</li>
	<li>Cut away the liver and stomach (on the rostral end of the small intestine), as well as the mesonephros and genital ridges if some are present.</li>
	<li>Isolate the small intestine. Again, be careful not to nip the intestine. From now on, the rosrrocaudal orientation of the gut tissue can be tracked using the sharp curvature present on the rostral end. Leave the small intestine in PBS at room temperature for as short a time as possible before embedding (see step 3.3).</li>
	<li>Finally, record the number of tail somites for each embryo to accurately determine the stage of embryonic development.</li>
</ol>
3. Sectioning of Embryonic Intestines
<ol>
	<li>Before proceeding with embryo dissections, melt 1.5 g agarose in 100 ml PBS (phosphate-buffered saline) and keep at 50 &#176;C.</li>
	<li>Pour melted agarose into an embedding mold (e.g. a closed 2 ml microcentrifuge tube that has been cut lengthwise to excise about 1/4 of the tube wall). Let the agarose cool down to approximately 42-45 &#176;C, it should be only slightly warm to the touch.</li>
	<li>Embed the embryonic intestine just before the agarose solidifies (this can be evaluated with forceps tips and will occur around 36-38 &#176;C). Holding the intestine with forceps by the folded rostral end, pull it very slowly through the agarose along the length of the mold. This helps to keep the gut straight while the agarose sets. Release the tissue as soon as it begins to resist being moved. Keep track of the rostral-caudal orientation of the gut.</li>
	<li>Put the mold in a refrigerator 2-3 minutes, to ensure the agarose has completely set.</li>
	<li>Take out the agarose from the mold (by sliding it out of the opened Eppendorf tube). With a blade, take out the excess agarose at both ends, making cuts perpendicular to the intestine.</li>
	<li>Glue the rostral end of the intestine/agarose block down on the metal stage of a microtome with vibrating blade. Trim the excess agarose on the sides of the intestine/agarose block.</li>
	<li>Mount the metal stage on the vibrating microtome chamber. If necessary, adjust the angle of the stage so the intestine is as vertical as possible (hence perpendicular to the blade). Cover the specimen with ice-cold PBS. Bring the microtome blade down to a few millimeters under the buffer surface.</li>
	<li>Make 200 &mu;m vibratome transverse cuts of the caudal-most small intestine, ensuring that each agarose slice contains a full intestinal section.</li>
</ol>
4. Culture of Intestinal Explants
<ol>
	<li>Gently deposit the freshly cut intestine/agarose slices flat onto the collagen gels with forceps, placing one slice towards the middle of each well.</li>
	<li>Incubate 3 days at 37 &#176;C, in a humid 5% CO2 atmosphere, to allow migration of NCC out of the explant.</li>
	<li>Take the intestine/agarose slices off the collagen gel very gently with forceps. Take care not to damage the gel below.</li>
	<li>Avoid touching the collagen gel directly during the subsequent incubation and wash steps in order to ensure that the cell migration pattern is not disturbed. Fix with 500 &#181;l of 4% PFA (paraformaldehyde) (in PBS) per well 1 hr at room temperature.</li>
	<li>Replace the fixative with 500 &#181;l of DAPI (4',6-diamidino-2-phenylindole) solution (5 &#181;g/ml in PBS) per well and incubate 10 min at room temperature.</li>
	<li>Wash each well 3x with 500 &#181;l of PBS for 5 min.</li>
	<li>Photograph the fluorescent cells (GFP and DAPI channels) embedded in the collagen gel within each well.</li>
</ol>
5. Image Analysis
We made extensive use of ImageJ17 to process and quantify the images generated after explant culture.
<ol>
	<li>Start by imaging a micrometer slide at the same magnification as the fluorescent cell photographs. Measure the length of one micron in pixels (using the Straight Line tool).</li>
	<li>Set the scale (Analyze/Set Scale; input number of pixels/micron).</li>
	<li>For each GFP fluorescence photograph, change the image format to 8-bit grayscale (Image/Type/8-bit).</li>
	<li>Adjust intensity of the signal (Image/Adjust/Brightness/Contrast).</li>
	<li>Subtract the background noise if necessary (Process/Subtract Background; adjust the rolling ball radius).</li>
	<li>Set a threshold to highlight cells and cell clumps (Image/Adjust/Threshold), then apply a watershed to divide the clumps (Process/Binary/Watershed).</li>
	<li>Analyze particles to specify regions of interest (ROI) and generate cell number statistics (Analyze/Analyze Particles; set size minimum to exclude remaining pixels).</li>
	<li>Set measurement options to include Feret&#39;s diameter (Analyze/Set Measurements).</li>
	<li>Group ROIs and measure as a whole to determine the Feret&#39;s diameter, an indication of cell spread (Analyze/Tools/ROI Manager/More/OR, then ROI Manager/Add, and ROI Manager/Measure).</li>
</ol>

<ol><li>Bronner, M. E., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+evolution+of+the+neural+crest%3A+An+overview%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Development and evolution of the neural crest: An overview.</a> Dev. Biol. 366 (1), 2-9 (2012).</li>
<li>Bergeron, K. F., Silversides, D. W., Pilon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+developmental+genetics+of+Hirschsprung%27s+disease%5BTitle%5D)+AND+%22Clin.+Genet%22%5BJournal%5D)">The developmental genetics of Hirschsprung's disease.</a> Clin. Genet. 83 (1), 15-22 (2013).</li>
<li>Obermayr, F., Hotta, R., Enomoto, H., Young, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+developmental+disorders+of+the+enteric+nervous+system%5BTitle%5D)+AND+%22Nat.+Rev.+Gastroenterol.+Hepatol%22%5BJournal%5D)">Development and developmental disorders of the enteric nervous system.</a> Nat. Rev. Gastroenterol. Hepatol. 10 (1), 43-57 (2012).</li>
<li>Sasselli, V., Pachnis, V., Bursn, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+enteric+nervous+system%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">The enteric nervous system.</a> Dev. Biol. 366 (1), 64-73 (2012).</li>
<li>Sanchez, M. P., Silos-Santiago, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Renal+agenesis+and+the+absence+of+enteric+neurons+in+mice+lacking+GDNF%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Renal agenesis and the absence of enteric neurons in mice lacking GDNF.</a> Nature. 382 (6586), 70-73 (1996).</li>
<li>Baynash, A. G., Hosoda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+endothelin-3+with+endothelin-B+receptor+is+essential+for+development+of+epidermal+melanocytes+and+enteric+neurons%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons.</a> Cell. 79 (7), 1277-1285 (1994).</li>
<li>Chalazonitis, A., Pham, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotrophin-3+is+required+for+the+survival-differentiation+of+subsets+of+developing+enteric+neurons%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons.</a> J. Neurosci. 21 (15), 5620-5636 (2001).</li>
<li>Goldstein, A. M., Brewer, K. C., Doyle, A. M., Nagy, N., Roberts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BMP+signaling+is+necessary+for+neural+crest+cell+migration+and+ganglion+formation+in+the+enteric+nervous+system%5BTitle%5D)+AND+%22Mech.+Dev%22%5BJournal%5D)">BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system.</a> Mech. Dev. 122 (6), 821-833 (2005).</li>
<li>Jiang, Y., Liu, M. T., Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Netrins+and+DCC+in+the+guidance+of+migrating+neural+crest-derived+cells+in+the+developing+bowel+and+pancreas%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Netrins and DCC in the guidance of migrating neural crest-derived cells in the developing bowel and pancreas.</a> Dev. Biol. 258 (2), 364-384 (2003).</li>
<li>Ramalho-Santos, M., Melton, D. A., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hedgehog+signals+regulate+multiple+aspects+of+gastrointestinal+development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Hedgehog signals regulate multiple aspects of gastrointestinal development.</a> Development. 127 (12), 2763-2772 (2000).</li>
<li>Natarajan, D., Marcos-Gutierrez, C., Pachnis, V., de Graaf, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Requirement+of+signaling+by+receptor+tyrosine+kinase+RET+for+the+directed+migration+of+enteric+nervous+system+progenitor+cells+during+mammalian+embryogenesis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Requirement of signaling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis.</a> Development. 129 (22), 5151-5160 (2002).</li>
<li>Young, H. M., Hearn, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GDNF+Is+a+chemoattractant+for+enteric+neural+cells%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">GDNF Is a chemoattractant for enteric neural cells.</a> Dev. Biol. 229 (2), 503-516 (2001).</li>
<li>Amiel, J., Sproat-Emison, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hirschsprung+disease%2C+associated+syndromes+and+genetics%3A+a+review%5BTitle%5D)+AND+%22J.+Med.+Genet%22%5BJournal%5D)">Hirschsprung disease, associated syndromes and genetics: a review.</a> J. Med. Genet. 45 (1), 1-14 (2008).</li>
<li>Pilon, N., Raiwet, D., Viger, R. S., Silversides, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+pre-+and+post-gastrulation+expression+of+Gata4+within+cells+of+the+inner+cell+mass+and+migratory+neural+crest+cells%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">Novel pre- and post-gastrulation expression of Gata4 within cells of the inner cell mass and migratory neural crest cells.</a> Dev. Dyn. 237 (4), 1133-1143 (2008).</li>
<li>Nagy, N., Goldstein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelin-3+regulates+neural+crest+cell+proliferation+and+differentiation+in+the+hindgut+enteric+nervous+system%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system.</a> Dev. Biol. 293 (1), 203-217 (2006).</li>
<li>Nagy, A., Gertsenstein, M., Vintersen, K., Behringer, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNagy%2C+A+%22Manipulating+the+mouse+embryo%3A+a+laboratory+manual%22">Manipulating the mouse embryo: a laboratory manual</a>. , 3rd ed, CSH Press. Cold Spring Harbor. 209-250 (2003).</li>
<li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NIH+Image+to+ImageJ%3A+25+years+of+image+analysis%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li>
<li>Byth, K. F., Thomas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AZD5438%2C+a+potent+oral+inhibitor+of+cyclin-dependent+kinases+1%2C+2%2C+and+9%2C+leads+to+pharmacodynamic+changes+and+potent+antitumor+effects+in+human+tumor+xenografts%5BTitle%5D)+AND+%22Mol.+Cancer+Ther%22%5BJournal%5D)">AZD5438, a potent oral inhibitor of cyclin-dependent kinases 1, 2, and 9, leads to pharmacodynamic changes and potent antitumor effects in human tumor xenografts.</a> Mol. Cancer Ther. 8 (7), 1856-1866 (2009).</li>
</ol>

The authors have nothing to disclose.

We show how our ex vivo explant culture technique can be used to precisely quantify enteric NCC migration potential in the presence of GDNF. Such precise quantification is greatly facilitated by using 200 &mu;m-thick vibratome gut sections instead of large pieces of approximate size, as previously described11,12,15. Indeed, this allows us to work with a reasonable number of cells in a highly reproducible setting. Of note, the uniform distribution of fluorescent enteric NCC within the caudal-most regio...

Neural crest cells (NCC) are a transient cell type unique to vertebrates that forms many derivatives during embryo development. This cell population arises at the border of the neural plate, adjacent to non-neural ectoderm1. During neurulation, bending of the neural plate places NCC along the dorsal edge of the forming neural tube. NCC then undergo an epithelial-mesenchymal transition, segregating and migrating away from the neural tube. NCC colonize various embryonic structures, including the digestive tract where they form the entire enteric nervous system (ENS), an interconnected network of neural ganglia embedded in the intestinal wall. As recently reviewed2,3, many genes have been involved in the development of this intricate structure.
Most of the ENS is derived from a small pool of NCC originating from the vagal neural tube (i.e. around the prospective hindbrain/spinal cord boundary)4. These neural progenitors reach the foregut around embryonic day (e) 9.0 in mice and then migrate caudally within the gut mesenchyme until approximately e15.0 to colonize the whole embryonic intestines. A minor subset of colonic neural progenitors is also provided by sacral NCC, which invade the posterior gut in the opposite direction up to the cecum4. Both vagal and sacral NCC require multiple migration-, proliferation-, survival- and differentiation-promoting cues to ensure complete formation of the ENS. In this regard, animal models - especially genetically modified mice - have been instrumental in the identification of several essential extracellular ligands: GDNF (glial cell-derived neurotrophic factor), Endothelin-3, Neurotrophin-3, BMPs (bone morphogenic proteins), Netrin, as well as Sonic and Indian Hedgehog (Shh and Ihh)5-10. Of these, GDNF signaling through the tyrosine kinase transmembrane receptor RET (Rearranged during transfection) is recognized as the most critical pathway for the attraction and guidance of NCC to and within the embryonic gut. GDNF is secreted by the gut mesenchyme and forms a spatiotemporally controlled rosrrocaudal gradient that is directly chemoattractive to enteric NCC, which express RET11,12.
Amongst other functions, the ENS regulates movement within the digestive tract through its interaction with smooth muscle in the intestinal wall. Absence of neural ganglia in the terminal region of the bowel results in Hirschsprung&#39;s disease: tonic contraction of the affected segment leads to blockage, upstream accumulation of digested material and massive distention of the gut and abdomen. Hirschsprung&#39;s disease occurs approximately one in 5,000 live births. The rostro-caudal migration pattern of enteric NCC is believed to be the main contributing factor to the etiology of Hirschsprung&#39;s disease. The colon, furthest from the source of migrating NCC and last portion of bowel to be colonized, is most susceptible to defects in ENS formation. In accordance with its crucial role in enteric NCC migration, disruption of GDNF/RET signaling is the main known genetic cause of Hirschsprung&#39;s disease13.
To better study NCC and ENS development, we generated a transgenic mouse line - named Gata4p[5kb]-GFP14 - in which migratory NCC are labeled with Green Fluorescent Protein (GFP). We next perfected an ex vivo cell migration assay, adapted from published work by other groups11,12,15, that now allows precise quantification of enteric NCC migration potential in the presence of various growth factors, such as GDNF.

<table><tbody><tr><td>DMEM powder</td><td>Wisent</td><td>219-010-XK</td><td>&amp;nbsp;</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Bioshop</td><td>SOB999</td><td>Biotechnology grade</td></tr><tr><td>Steriflip vacuum filtration system (0.22 micron)</td><td>EMD Millipore</td><td>SCGP00525</td><td>&amp;nbsp;</td></tr><tr><td>Penicilin-Streptomycin solution, 100x</td><td>Wisent</td><td>450-201-EL</td><td>&amp;nbsp;</td></tr><tr><td>Fetal bovine serum</td><td>Wisent</td><td>095-150</td><td>High quality grade</td></tr><tr><td>Collagen I</td><td>BD biosciences</td><td>354236</td><td>&amp;nbsp;</td></tr><tr><td>NaOH</td><td>Bioshop</td><td>SHY700</td><td>Diluted from 10 N stock then sterile-filtered</td></tr><tr><td>GDNF</td><td>Cedarlane</td><td>CLCYT305</td><td>&amp;nbsp;</td></tr><tr><td>Falcon 24-well Plate</td><td>BD biosciences</td><td>353047</td><td>&amp;nbsp;</td></tr><tr><td>Dissecting scissors</td><td>Fisher Scientific</td><td>089515</td><td>&amp;nbsp;</td></tr><tr><td>Glass Petri dish</td><td>VWR</td><td>89000-306</td><td>&amp;nbsp;</td></tr><tr><td>PBS</td><td>Sigma</td><td>P5493</td><td>Cell culture grade</td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>M125</td><td>&amp;nbsp;</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-20</td><td>&amp;nbsp;</td></tr><tr><td>Agarose</td><td>Bioshop</td><td>AGA001</td><td>Biotechnology grade</td></tr><tr><td>Surgical blade</td><td>Feather</td><td>21</td><td>&amp;nbsp;</td></tr><tr><td>All Purpose Instant Krazy Glue Pen</td><td>Krazy Glue</td><td>KG824</td><td>&amp;nbsp;</td></tr><tr><td>HM 650V Vibrating-Blade Microtome</td><td>Thermo Scientific</td><td>920110</td><td>&amp;nbsp;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>&amp;nbsp;</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9564</td><td>&amp;nbsp;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Table of specific reagents and equipment.&lt;/p&gt;</td></tr></tbody></table>

a quantitative cell migration assay for murine enteric neural progenitors

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

The following results are representative of what can be obtained with the technique described here (Figure 1). The use of growth factors (i.e. GDNF) stimulates migration of GFP-expressing enteric NCC out of the intestinal explant and into the collagen gel (Figure 2). Though some cells come out of the explant in the absence of growth factors, these are mostly not GFP-labeled and represent passive entry. It is necessary to remove the intestinal slice from the collagen gel in order...

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

The overall goal of this procedure is to quantify the migration potential of enteric neuro progenitors in the presence of various growth factors. This is accomplished by first dissecting mouse embryonic, small intestines populated by fluorescent neuro crust cells. The second step is to use a vibrating microtome to make 200 micron thick slices of intestines.Next, these slices are incubated on top of a collagen gel containing growth factors. The final step is to peel off the slices in order to reveal the migrating cells underneath. Ultimately, fluorescence microscopy is used to show the pattern of migrating cells within the collagen gel.This method can help answer a key question in developmental biology, such as providing a better understanding of the interplay between various signaling pathways underlying the formation of the peripheral nervous system. 12 days after a vaginal plug is found, euthanize the female mouse and remove the uterus, placed the uterus into a glass petri dish filled with ice. Cold PBS cut the uterus transversely between individual deum swellings to isolate each embryo implantation site under a dissecting microscope, use fine forceps to remove the muscle layer of the uterus.Open the visceral yolk sack and amnion to reveal the embryo. Take care when severing the blood vessels, joining the embryo to the yolk sack as they're intertwined with the developing intestines. Next, insert closed forceps into the abdominal cavity just above the dark red colored liver and allow them to open.This creates a transversal opening in the abdomen cavity. Pull the opened forceps down towards the posterior end of the embryo to open the abdomen completely. Once opened, grab the connective tissue behind the liver and pull the guts out.Taking care not to break the intestines. Cut the colon at any location to free the guts from the rest of the embryo. Reserve the tail portion for later.Tease out the connective tissue from the cecum and then the intestines working carefully not to wound them on the rostral end of the small intestine. Cut away the liver and stomach as well as the meso, nephros and genital ridges if present. Finally, isolate the small intestine by cutting away the cecum and colon as well as the remaining connective tissue.As before, take care not to nick the intestine. Leave the small intestine in PBS at room temperature for as short a time as possible before embedding. After severing the embryo at the neck, record the number of tail somites to accurately determine the stage of embryonic development.Before proceeding with embryo dissections, melt 1.5 grams of agros in 100 milliliters of PBS and keep at 50 degrees Celsius. Prepare the embedding mold by cutting a two milliliter micro centrifuge tube lengthwise to excise about one quarter of the tube wall. Pour the melted agros into the mold and let it cool down until it is only slightly warm to the touch.When ready, hold the intestine with forceps by the folded rostral end and pull it very slowly through the agros, along the length of the mold. This helps to keep the gut straight while the agro sets release the tissue as soon as it begins to resist being moved and keep track of the rostral coddle orientation of the gut. Next, place the mold in a refrigerator for two to three minutes to set completely.Once set, remove the agros from the mold and trim the excess agros at both ends, making cuts perpendicular to the intestine. Glue the rostral end of the agros block to the metal stage of a microtome and trim the excess aros on all sides. Next, mount the metal stage on the vibrating microtome chamber.If necessary, adjust the angle of the stage so the intestine is as vertical as possible. Cover the specimen with ice cold PBS before bringing the microtone blade down to a few millimeters under the buffer surface. When ready, make 200 micron transverse cuts of the coddle most small intestine.Ensuring that each agro slice contains a full intestinal section. Gently deposit the freshly cut slices onto a previously prepared collagen gel. Placing one slice towards the middle of each well to allow the migration of neuro crest cells out out of the eggplants, incubate the slices for three days.After this time, take the slices off the collagen gel gently with forceps taking care not to damage the gel below. To ensure that the cell migration pattern is not disturbed, it is important to avoid touching the collagen gel directly during the subsequent incubation and wash steps. First, fix the cells with 500 microliters of 4%PFA per well for one hour at room temperature.Next, replace the fixative with 500 microliters of DPI solution per well and incubate for 10 minutes. At room temperature, carefully remove the DPI solution and wash each well three times with 500 microliters of PBS for five minutes. Lastly, photograph the fluorescent cells embedded in the collagen gel within each well.In this example, growth factors were used to stimulate the migration of GFP expressing enteric neuro crust cells. The dotted line represents the approximate size and location of the explan. Before it was taken off the gel, the number and spread of GFP expressing cells migrating out of intestinal explan was found to be significantly greater in treated versus untreated conditions.After watching this video, you should have a good understanding of how to determine the migration potential of enteric neural progenitors by incubating intestinal slices over a collagen gel containing growth factors.

a-quantitative-cell-migration-assay-for-murine-enteric-neural

Research

JoVE Journal

Neuroscience

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

This video demonstrates the neural precursor cell neurosphere migration assay—an in vitro assay to study the migratory response of neural precursor cells from the neurosphere—via microscopic analysis.

NPC Neurosphere Assay: An In Vitro Assay to Assess the Migratory Response of Neural Precursor Cells from the Neurosphere

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

Analyzing Murine Schwann Cell Development Along Growing Axons

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

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.

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

Learn a quantitative cell migration assay for murine enteric neural progenitors using fluorescence microscopy and collagen gel incubation. This method enables researchers to study growth factor effects on neural crest cell migration.

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Explore More Videos