Name: a quantitative cell migration assay for murine enteric neural progenitors
Uploaded: 2013-09-18T15:15:00
Description: Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

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 ...

<ol>
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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 border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</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></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></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></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></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></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></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></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></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

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 ...

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