Fig. 1. Foetal gut-conditioned medium (FGCM) and GDNF promote invasion of collagen gels by ENCCs and enteric axons. (A) E11.0-11.5 proximal small intestine explants from wild-type (a-h) or Ret.k— (i,j) mouse embryos were cultured either in control medium (CM; a,b,e,f) or in the presence of 50% FGCM (c,d) or 10 ng/ml rGDNF (g-j) in three-dimensional collagen matrices. At the end of the culture period, explants were stained with PGP9.5 (a,c,e,g,i) and Tuj-1 (b,d,f,h,j) to visualise ENCCs and enteric axons. Both FGCM and rGDNF promote the invasion of collagen gels by NC-derived cells and axons (compare a,b with c,d and e,f with g,h). Explants derived from Ret.k— embryos are devoid of ENCCs or axons. (B) The PENCC-containing postbranchial region ventral to the dorsal aorta (boxed in part k; shows an E9.0 mouse embryo hybridised with a Ret-specific riboprobe) was dissected from wild-type E9.0 mouse embryos and cultured in control medium (CM; 1) or in control medium supplemented with 10 ng/ml of rGDNF (CM+GDNF; m). At the end of the culture period, explants were stained for RET (green in l,m) and counterstained for DAPI (blue in inset of part m). rGDNF promotes the invasion of the collagen gel by explant-derived cells. The vast majority of emigrating cells express RET (inset in part m), indicating that they are of NC origin. Broken line indicates the boundary between the collagen gel matrix and the explant. (C) E11.0-11.5 proximal small intestine explants from wild-type (n-q) or miRet⁵¹ (r,s) embryos were co-cultured with either control COS-7 cells (COS; n,o) or COS-7 cells expressing GDNF (COS/GDNF; p-s). In every panel, the small intestine explant is on the right (left border highlighted by a broken line) and the COS-7 cells on the left (indicated by broken line). At the end of the culture period, explants were stained for RET (n,p,r) and Tuj1 (o,q,s) and counterstained with DAPI (inset in n,p). COS/GDNF cells induce cell and axonal migration always from the side of the explants facing the transfected cells (p,q). Insets in n,p show the area between explants and COS cells viewed with DAPI filter. The presence of a large number of nuclei specifically in the inset of p indicates that, in addition to axons, a large number of cells originating in the small intestine explant have invaded the collagen gel in response to GDNF. Explants from Ret.k⁵¹ homozygous embryos, cultured and processed in parallel to wild-type ones, showed reproducibly a weaker response.

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