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First published online 2 October 2008
doi: 10.1242/dev.022319

Development 135, 3555-3565 (2008)
Published by The Company of Biologists 2008
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Notch signaling is required for the maintenance of enteric neural crest progenitors

Yoshiaki Okamura1 and Yumiko Saga1,2,*

1 Department of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
2 Division of Mammalian Development, National Institute of Genetics, Yata 1111, Mishima 411-8540, Japan.


Figure 1
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  		Fig. 1. Defects of the NCC-specific Pofut1-knockout mouse. (A) External morphology of control and Pofut1 cKO mice at postnatal day 1 (P1). The Pofut1 cKO mouse is relatively small, but otherwise shows no apparent morphological defects. Arrowheads indicate the stomach. (B-E) Immunostaining of the gastrointestinal tract at E16.5 in control (B,C) and Pofut1 cKO (D,E) embryos with the anti-neurofilament antibody 2H3.

 

Figure 2
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  		Fig. 2. Lineage analysis of ENCCs. Enteric neural crest lineages were traced using β-galactosidase activity from E10.5 to E13.5 in control (A-D) and Pofut1cKO (E-H) mouse embryos by crossing Pofut1+/-;Wnt1-Cre and Pofut1flox/flox;R26R mice. The gastrointestinal tracts are outlined (dashed lines). Arrowheads (A,E) indicate migratory wavefront of ENCCs. st, stomach; ca, cecum.

 

Figure 3
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  		Fig. 3. Detection of apoptosis and proliferation in ENCCs. (A-H) Immunohistological analysis of control (A-D) and Pofut1 cKO (E-H) mouse embryos using anti-caspase 3 (red) and anti-GFP (green) antibodies to visualize apoptotic cells and ENCCs, respectively, at E11.5 (A,B,E,F) and E12.5 (C,D,G,H). (I-P) Immunohistological analysis of control (I-L) and Pofut1 cKO (M-P) embryos using anti-BrdU (red) and anti-GFP (green) antibodies to visualize proliferating cells and ENCCs, respectively, at E11.5 (I,J,,M,N) and E12 (K,L,O,P). Arrowheads indicate proliferating ENCCs. (Q,R) Quantitation of ENCC proliferation at E11.5 (Q) and E12.5 (R). Average percentages of BrdU-incorporating ENCCs are shown with s.d. ns, not significant.

 

Figure 4
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  		Fig. 4. Neuronal and glial differentiation of ENCCs. Immunohistochemical analysis of control (A-C,G) and Pofut1 cKO (D-F,H) mouse embryos with anti-TuJ1 (red) and anti-GFP (green) antibodies to visualize neuronal cells and ENCCs, respectively, at E10.5-12.5 (A-F), or with anti-BFABP (magenta) and anti-GFP (green) antibodies to visualize glial progenitors and ENCCs, respectively, at E11.5 (G,H). Arrowheads (G,H) indicate BFABP-positive ENCCs. (I,J) Quantitation of neuronal differentiation at E10.5 (I) and E11.5 (J). Average percentages of TuJ1-positive ENCCs are shown with s.d. (K) Quantitation of glial differentiation at E11.5. Average percentages of BFABP-positive ENCCs are shown with s.d.

 

Figure 5
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  		Fig. 5. Downregulation of Sox10 in Pofut1-null ENCCs. (A-H) Immunohistological detection of Sox10 (red) expression within ENCCs (green) in control (A-D) and Pofut1 cKO (E-H) mouse embryos at E10.5 (A,B,E,F) and E11.5 (C,D,G,H). (I,J) Quantitation of Sox10-positive cells at E10.5 (I) and E11.5 (J). Average percentages of Sox10-positive ENCC cells are shown with s.d. (K) Quantification of the proliferation rate in Sox10-positive ENCCs at E11.5. Average percentages of BrdU incorporation in proliferating Sox10-positive ENCC cells are shown with s.d. ns, not significant.

 

Figure 6
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  		Fig. 6. Expression of Notch signaling genes in the mouse gastrointestinal tract. In situ hybridizations were performed using RNA probes against Notch1, Notch4, Dll1, Dll3, Dll4 and Jag1 (magenta), together with the immunohistological detection of NCCs using an anti-p75NTR antibody (green) in E11.5 stomach and intestine. Open arrowheads indicate expression in ENCCs, and solid arrowheads indicate expression in neighboring cells. The boxed regions are shown at high magnification in the right-hand panel of each pair.

 

Figure 7
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  		Fig. 7. ENCCs expressing Hes1 are decreased, whereas those expressing Mash1 are increased, in the Pofut1 cKO embryo. Immunohistological analysis of control (A-C,G-I) and Pofut1 cKO (D-F,J-L) mouse embryos at E10.5 using antibodies against Hes1 (A,C,D,E, magenta), Mash1 (G,I,J,L, magenta) and GFP (B,C,E,F,H,I,K,L, green). Arrowheads indicate Hes1-positive or Mash1-positive ENCCs in control (C,I) and Pofut1 cKO (F,L) embryos. (M,N) Quantification of Hes1-positive ENCCs (M) and Mash1-positive ENCCs (N) at E10.5. Average percentages of the total ENCC cell population expressing each gene are shown with s.d.

 

Figure 8
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  		Fig. 8. Comparative analysis of ENCCs expressing Mash1 and Sox10. Immunohistological analysis of control (A-C) and Pofut1 cKO (D-F) mouse embryos at E10.5 using antibodies against Mash1 (A,C,D,F, green) and Sox10 (B,C,E,F, red). Arrowheads indicate Mash1 single-positive ENCCs, and arrows indicate Sox10 single-positive ENCCs. (G) Statistical analyses of Mash1-single positive, Sox10-single positive or Mash1/Sox10 double-positive cells at E10.5. Average percentages of the ENCC progenitor population expressing Mash1 or Sox10 are shown with s.d.

 

Figure 9
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  		Fig. 9. A hypothetical model for the function of Notch signaling during mouse ENS development. The NCCs begin to express Sox10 when they detach from the neural tube at around E8.5. After arriving at the foregut (E9.5), a subset of the NCCs begins to express Mash1. In turn, Mash1 suppresses Sox10 expression, and Mash1-positive cells then eventually produce neurons. The remaining NCCs continue to express Sox10 to maintain the pool of ENS progenitors. Notch signaling might be required for the continuous expression of Sox10 by suppressing Mash1. TC cells, transient catecholaminergic cells (see Discussion).

 

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