Abstract
Hirschsprung disease (HSCR), also named aganglionic megacolon, is a severe congenital malformation characterized by a lack of enteric nervous system (ENS) in the terminal regions of the bowel (Bergeron et al., 2013). As the ENS notably regulates motility in the whole gastrointestinal track, the segment without neurons remains tonically contracted, resulting in functional intestinal obstruction and accumulation of fecal material (megacolon). HSCR occurs when enteric neural progenitors of vagal neural crest origin fail to fully colonize the developing intestines. These “enteric” neural crest cells (ENCCs) have to migrate in a rostro-caudal direction during a fixed temporal window, which is between embryonic day (e) 9.5 and e14.5 in the mouse (Obermayr et al., 2013). Recently, our group generated a new HSCR mouse model called Holstein in which migration of ENCCs is impaired because of increased collagen VI levels in their microenvironment (Soret et al., 2015). Here, we describe the method that allowed us to demonstrate the cell-autonomous nature of this migration defect. In this system adapted from a previously described heterotopic grafting approach (Breau et al., 2006), the donor tissue is a fully colonized segment of e12.5 midgut while the host tissue is an aneural segment of e12.5 hindgut. Extent of ENCC migration in host tissue is assessed after 24 h of culture and is greatly facilitated when donor tissue has a transgenic background such as the Gata4-RFP (Pilon et al., 2008) that allows endogenous labeling of ENCCs with fluorescence. Depending of the genetic background of donor and host tissues, this approach can allow evaluating both cell-autonomous and non-cell-autonomous defects of ENCC migration.
Keywords: Enteric nervous system, Mouse, Neural crest cells, Cell migration, ex vivo
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 1. Key steps of the dissection procedure. Step 1. With fine forceps, remove the uterine muscle layers. Step 2. Open the extra-embryonic membrane to access the embryo, cut the blood vessels connecting the embryo to the placenta/extra-embryonic membranes. Step 3. Cut the embryo head and open the abdominal cavity. Step 4. Pull the whole gastrointestinal tract out of the abdominal cavity. Step 5. Remove the liver, cut the esophagus and release the digestive tract of the connective tissue. Step 6. Free the gastrointestinal tract by cutting it at the anus. Figure 2. Overview of graft assembly and representative result. A. Schematic representation of a heterotopic e12.5 midgut-hindgut graft onto a nitrocellulose filter in a chamber of an 8-chamber slide. Red dots in midgut tissue represent ENCCs labeled by RFP (DsRed2) fluorescence provided by the G4-RFP transgene; B. Representative images of a graft (delineated by dotted lines) after 24 h of culture, showing the colonization of a previously aneural hindgut host tissue by fluorescently labeled ENCCs from midgut donor tissue. Pictures were taken using an Infinity-2 camera mounted on a Leica M205FA fluorescent stereomicroscope and images were analyzed using the ImageJ software. The white arrow points to the location of the migration front at the end of the culture period. Scale bar: 150 μm.
Notes
It is noteworthy that this method is greatly simplified when donor tissues are taken from embryos bearing a transgene such as the G4-RFP transgene (Pilon et al., 2008) that labels ENCCs with fluorescence. At step 10, this can allow the identification of mutant tissues (i.e., displaying delayed migration) by simple fluorescent microscopy instead of having to wait for genotyping results after graft assembly. Moreover, at step 13, such an intrinsic fluorescent labeling greatly facilitates the analysis of ENCC migration which otherwise requires immunofluorescence labeling using an antibody against a marker of undifferentiated enteric neural progenitors such as Sox10.
Acknowledgments
The Pilon laboratory is funded by grants from the Canadian Institute of Health Research (CIHR), the Natural Science and Engineering Research Council of Canada (NSERC) and the Fondation du grand défi Pierre Lavoie. RS holds a fellowship from the Fonds de la recherche du Québec Santé (FRQS) whereas NP is a FRQS Junior 2 Research Scholar as well as the recipient of a UQAM Research Chair on Rare Genetic Diseases. The authors thank the innovative work performed by Breau et al. (2006), on which this protocol was based.
References
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