Fig. 3. Endogenous GDNF is a chemoattractant for ENC cells. Segments of small intestine from E10.5 wild-type embryos were cultured either alone (A-D) or with caecum from E11.0 wild-type (E-H,M-P) or GDNF-deficient (I-L) embryos. In M-P, explants were cultured in the presence of GDNF blocking antibodies. At the end of the culture period, explants were stained for RET and counterstained with DAPI. (A,E,I,M) Bright-field images of the explants; (C,G,K,O) corresponding fluorescent images; (B,D,F,H,J,L,N,P) higher magnification fluorescent images of the left (B,F,J,N) and right (D,H,L,P) ends of the explants. The position of the small intestine explants relative to the caecum was random and unrelated to the original rostrocaudal polarity of the tissue. To quantitate the effect of caecum under the various culture conditions, we counted the RET-expressing cells present outside the borders of the small intestine explant in the semicircles defined by the rounded ends of the explant (shown schematically by the broken line in A). The average number of cells recorded at each end of the small intestine explant is shown in the corresponding high magnification images. Note the large number of cells filling the space between small intestine and wild-type caecum cultured in control medium (F). The inset in this panel shows overlaid fluorescent images taken with the FITC and DAPI filters. The majority of cells emigrating from the small intestine explant express RET, indicating that they are of NC origin. The number of RET+ cells emigrating from small intestine towards GDNF-deficient caecum (J) or towards wild-type caecum in the presence of anti-GDNF blocking antibodies (N) is significantly reduced.

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