A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Summary

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.

Abstract

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.

Introduction

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 revi....

Protocol

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 (Comité 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.

1. Prepare complete 5x DMEM (Eagle's modified essential medium) including standard antib....

Results

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.......

Discussion

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 μm-thick vibratome gut sections instead of large pieces of approximate size, as previously described^11,12,15. 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM powder	Wisent	219-010-XK
NaHCO3	Bioshop	SOB999	Biotechnology grade
Steriflip vacuum filtration system (0.22 micron)	EMD Millipore	SCGP00525
Penicilin-Streptomycin solution, 100x	Wisent	450-201-EL
Fetal bovine serum	Wisent	095-150	High quality grade
Collagen I	BD biosciences	354236
NaOH	Bioshop	SHY700	Diluted from 10 N stock then sterile-filtered
GDNF	Cedarlane	CLCYT305
Falcon 24-well Plate	BD biosciences	353047
Dissecting scissors	Fisher Scientific	089515
Glass Petri dish	VWR	89000-306
PBS	Sigma	P5493	Cell culture grade
Dissecting microscope	Leica	M125
Dumont #5 Forceps	Fine Science Tools	11251-20
Agarose	Bioshop	AGA001	Biotechnology grade
Surgical blade	Feather	21
All Purpose Instant Krazy Glue Pen	Krazy Glue	KG824
HM 650V Vibrating-Blade Microtome	Thermo Scientific	920110
Paraformaldehyde	Sigma-Aldrich	P6148
DAPI	Sigma-Aldrich	D9564
Table of specific reagents and equipment.