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First published online 19 April 2006
doi: 10.1242/dev.02375

Development 133, 2075-2086 (2006)
Published by The Company of Biologists 2006
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Maintenance of mammalian enteric nervous system progenitors by SOX10 and endothelin 3 signalling

Nadege Bondurand*,{dagger}, Dipa Natarajan*, Amanda Barlow*,{ddagger}, Nikhil Thapar§ and Vassilis Pachnis

Division of Molecular Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.


Figure 1
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  		Fig. 1. Overexpression of SOX10 inhibits overt neuronal and glial differentiation of EPCs. (A,B) Staining of wild-type EPC colonies expressing either GFP (A) or SOX10-GFP (B) transgenes with antibodies for GFP (green) and TuJ1 (red). (C) Percentage of TuJ1+ cells present in GFP- (green bars) or SOX10-GFP-(blue bars) expressing EPC colonies 5, 7 and 10 days after plating. (D-K) Day 5 EPC colonies expressing GFP (D,F,H,J) or SOX10-GFP (E,G,I,K) transgenes were immunostained for GFP (D,E,H,I), MASH1 (F,G) or RET (J,K). Optic fields in D,E and H,I are the same as those shown in F,G and J,K, respectively. (L,M) Day 10 GFP or SOX10-GFP colonies double immunostained for GFP (green) and GFAP (red).

 

Figure 2
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  		Fig. 2. EDN3 signalling inhibits lineage commitment and differentiation of EPCs. Wild-type EPCs were isolated from cultures of embryonic (A-D,H-K,L-O,R,S) or postnatal (E,F,P,Q,T,U) gut and maintained in either standard (control) medium (CM; A,C,E,H,J,L,N,P,R,T) or CM supplemented with 10 nM EDN3 (B,D,F,I,K,M,O,Q,S,U). Colonies were fixed 5 (A,B,H-K,L,M) or 10 (C-F,N-Q,R-U) days after plating and immunostained for TuJ1 (A-F), MASH1 (H,I), RET (J,K), B-FABP (L,M), GFAP (N-Q) and SOX10 (R-U). In all panels, colonies were counterstained with DAPI. (G) Percentage of TuJ1+ cells in colonies maintained either in standard medium (CM; yellow bars) or medium supplemented with 10 nM EDN3 (CM+EDN3; green bars) 5, 7 and 10 days after plating. (V) Percentage of SOX10+ cells in colonies maintained in CM (yellow bars) or CM+EDN3 (green bars) 1, 5 or 10 days after plating.

 

Figure 3
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  		Fig. 3. The inhibitory effect of EDN3 on lineage commitment and differentiation of EPCs is reversible. Colonies from embryonic EPCs (A,B,D,E) were maintained in standard medium (CM; A,D) or standard medium supplemented with 10 nM EDN3 (CM+EDN3; B,E) and immunostained for MITF (A,B) or SMA (D,E). (C,F) Images of cultured SkMe15 cells known to express MITF [American Type Culture Collection, ATCC (Du et al., 2003Go)] (C) and a cell in a primary embryonic gut culture that expresses SMA (F). (G) The culture conditions of freshly isolated (day 0) EPCs. At the end of the culture period (day 12) colonies in each condition were fixed and immunostained for TuJ1 (H,J,L,N) or GFAP (I,K,M,O). All colonies were counterstained with DAPI.

 

Figure 4
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  		Fig. 4. Normal SOX10 activity and EDN3 signalling are required for formation of NLBs and EPCs. Guts from E11.5 wild-type (A), Sox10+/LacZ (B) or Ret51/51 (C) embryos were dissociated and cultured under conditions that promote the formation of NLBs. After fixation, cultures were immunostained for TuJ1 (red) and GFAP (green), and counterstained with DAPI. NLBs form readily in gut cultures from wild-type and Ret51/51 embryos but fail to form in cultures from embryos hemizygous for Sox10. In wild-type gut cultures maintained in standard medium (CM), NLBs form characteristic colonies composed of undifferentiated cells, neurons and glia (Bondurand et al., 2003Go). Such a colony immunostained for TuJ1 (red) and GFAP (green) is shown (D). These characteristic colonies do not appear in the presence of BQ788 (E). In the absence of CEE, embryo gut cultures failed to form NLBs (F). However, addition of EDN3 was sufficient to rescue the formation of NLBs (G) and EPCs capable of generating multilineage colonies. Part of such an EPC colony is shown in the inset of G.

 

Figure 5
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  		Fig. 5. Reduction of the pool of ENS progenitors in Edn3ls/ls embryos. (A) Whole-mount preparation of gut from Edn3+/+; TgWnt1Cre; R26YFPStop embryos immunostained for YFP. Box indicates the part of gut used to generate the intestinal acute cultures. (B) Bars represent the fraction of double positive (SOX10+YFP+) cells relative to the total population of neural crest-derived (YFP+) cells in acute intestinal cultures established from embryos wild-type (red) or heterozygous (blue) and homozygous for Edn3ls (green).

 

Figure 6
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  		Fig. 6. Reduced EDN3 signalling decreases the proliferation of specific subpopulations of enteric neural crest cells. (A) Whole-mount preparation of gut from Edn3+/+; TgWnt1Cre; R26YFPStop embryos immunostained for YFP. Box 1 indicates the part of gut used to generate the intestinal acute cultures examined in C and D. Box 2 indicates the area of the gut analysed in B and E. (B) Confocal image of an embryonic gut region corresponding to box 2 of A. Gut was immunostained for BrdU (red) and YFP (green). (C) Bars represent the fraction of BrdU+YFP+ cells relative to the total population of neural crest-derived (YFP+) cells in acute intestinal cultures from wild-type (red), Edn3+/ls (blue) and Edn3ls/ls (green) embryos. (D) Percentage of BrdU+SOX10+ cells within the total population of SOX10+ cells in acute intestinal cultures from wild-type, Edn3+/ls and Edn3ls/ls embryos. (E) Fraction of BrdU+YFP+ cells relative to the total population of YFP+ cells at the migratory wavefront of enteric neural crest cells.

 

Figure 7
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  		Fig. 7. Increased neuronal differentiation of enteric neural crest cells at the migratory front. (A,B) Whole-mount preparations of guts from E11.5 TgWnt1Cre;R26YFPStop embryos wild-type (Edn3+/+; A) or homozygous for the ls allele (Edn3ls/ls; B); guts were immunostained for YFP (green) and TuJ1 (red). Images correspond to the front of enteric neural crest cell migration (part of box 2 of the gut shown in Fig. 6A). (C) Bars represent the fraction of double-positive (TuJ1+YFP+) cells within the total population of YFP+ cells present at the front of migration of wild-type (red), Edn3+/ls (blue) and Edn3ls/ls (green) embryos.

 

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© The Company of Biologists Ltd 2006