QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Natarajan, D. Articles by de Graaff, E. Search for Related Content PubMed PubMed Citation Articles by Natarajan, D. Articles by de Graaff, E.

Requirement of signalling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis

Dipa Natarajan, Camelia Marcos-Gutierrez^*, Vassilis Pachnis and Esther de Graaff

Division of Molecular Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
^* Present address: GlaxoSmithKline Biologicals, Rue de l'Institut 89, B-1330 Rixensart, Belgium
Present addresses: Department of Clinical Genetics, Erasmus University, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands

View larger version (43K): [in a new window]   Fig. 5. Activation of the MAPK and PI(3)K signalling pathways is necessary for the chemoattractant response of ENCCs and enteric axons to GDNF. (A) Segments of proximal small intestine dissected from E11.0 mouse embryos were co-cultured with a clump of COS/GDNF cells (shown to the right of small intestine explants in a,c,e), in either control medium (a,b), or in medium supplemented with PI(3)K inhibitor (LY294002; c,d) or MEK1 inhibitor (PD98059; e,f). At the end of the experiment, cultures were stained for RET (a,c,e) and Tuj1 (b,d,f) and counterstained for DAPI. Reduced cell and axonal emigration were observed in the presence of inhibitors. (B) To examine whether the presence of the inhibitors resulted in increased apoptotic death, similar small intestine explants were cultured in control medium (left panel), or medium supplemented with 30 µM LY29002 (middle panel) or 60 µM PD98059 (right panel). At the end of the culture period, sections from the explants were stained for TUNEL to identify apoptotic cells. Note that under the present culture conditions, neither LY29002 nor PD98059 increases significantly the number of TUNEL+ nuclei. (C) To quantify the effect of increasing concentrations of PI(3)K and MEK1 inhibitors on ENCC migration, the number of RET-expressing cells present between the small intestine segment and COS/GDNF cells was counted. The response of explants cultured in control medium was considered as 100%. Increasing concentrations of PI(3)K inhibitor result in complete abrogation of the response. By contrast, a residual but significant response was observed even at high concentrations of MEK1 inhibitor. Nine explants were analysed for all concentrations tested for each inhibitor. Four explants were analysed in the experiments where the PI(3)K and MEK1 inhibitors were combined. The concentrations of LY294002 and PD98059 used for all explants shown in this figure were 10 µM and 30 µM, respectively.

View larger version (103K): [in a new window]   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.

View larger version (73K): [in a new window]   Fig. 2. Expression of Gdnf and Ret during mouse gut organogenesis. Wild-type (A-C), Ret.k— (D) embryos and gut preparations (E-I), were stained by whole-mount in situ hybridisation histochemistry using riboprobes specific for Gdnf (A,B,D,E,F,H) or Ret (C,G,I). In E9.0-9.5 embryos, Gdnf mRNA is detected in the splachnic mesenchyme of the stomach (indicated by arrow in all panels of the top row and by s in all panels of the bottom row) and in the branchial arches (black arrowheads in A,B). At this stage, RET-expressing ENS progenitors (white arrowhead in C) are starting to invade the GDNF-expressing region of the foregut (C; outline of the stomach is indicated by line and an arrow). (E) In the gastrointestinal tract of E9.5 embryos, expression of Gdnf is highest in the stomach region. However, in the gut of E10.5 embryos (F), the main site of Gdnf expression has shifted to the caecum (indicated by c). At this stage, the front of migrating NC cells was positioned rostrally to the high Gdnf-expressing caecum. At later stages, the domain of Gdnf expression in the hindgut extends posteriorly along with the wave of migrating NC cells (white arrows in H,I). Note the similar pattern of expression of Gdnf in RET-deficient mouse embryos (D). In addition to the gastrointestinal tract, Gdnf is also expressed in the pharyngeal pouches of the branchial arches (black arrowheads in A,B) and in a small region ventrally to the dorsal aortas (white arrowhead in A,B), the site where the progenitors of the superior cervical ganglia will first coalesce.

View larger version (97K): [in a new window]   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.

View larger version (85K): [in a new window]   Fig. 4. Abnormal migration of ENS progenitor cells in embryos homozygous for mutant alleles of Ret. (A-D) E9.5 wild-type (A) and Ret.k— (B) embryos and guts dissected from E10.5 wild-type (C) and miRet51 (D) embryos were hybridised as whole-mount preparations with a Sox10 cRNA probe. In E9.5 wild-type embryos (A), Sox10+ ENS progenitors were detected in the postbranchial region (black arrowhead) and in the foregut (white arrowhead). By contrast, in similar stage Ret.k— embryos (shown in B), no Sox10+ cells were present in the foregut despite the presence of such cells in the immediate postbranchial region (black arrowhead). At this stage, both wild-type and mutant embryos express high levels of Sox10 in cranial ganglia (cg) and in dorsal root ganglia (drg). In the gut of wild-type E10.5 embryos (C), the front of migrating Sox10+ cells (black arrow) has passed the midpoint of the midgut (halfway between the caudal region of the pancreas and the caecum). By contrast, in the gut of miRet51 homozygous embryos (D), the front of the migrating cells (black arrow) was located more rostrally, in the beginning of the midgut (black arrow). p, pancreas; ce, caecum; co, colon (hindgut).