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Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut.

Fu M, Lui VC, Sham MH, Pachnis V, Tam PK - J. Cell Biol. (2004)

Bottom Line: The pro-neurogenic effect of glial cell line--derived neurotrophic factor (GDNF) on NCCs was abolished by Shh.In gut explants, NCCs migrated from the explants onto the adjacent substratum if GDNF was added, whereas addition of Shh abolished this migration.Our data suggest that Shh controls the proliferation and differentiation of NCCs and modulates the responsiveness of NCCs toward GDNF inductions.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, The University of Hong Kong, 21 Sassoon Rd., Pokfulam, Hong Kong, HKSAR China.

ABSTRACT
Enteric neural crest cells (NCCs) migrate and colonize the entire gut and proliferate and differentiate into neurons and glia of the enteric nervous system in vertebrate embryos. We have investigated the mitogenic and morphogenic functions of Sonic hedgehog (Shh) on enteric NCCs in cell and organ culture. Enteric NCCs expressed Shh receptor Patched and transcripts encoding the Shh signal transducer (Gli1). Shh promoted the proliferation and inhibited the differentiation of NCCs. The pro-neurogenic effect of glial cell line--derived neurotrophic factor (GDNF) on NCCs was abolished by Shh. In gut explants, NCCs migrated from the explants onto the adjacent substratum if GDNF was added, whereas addition of Shh abolished this migration. Neuronal differentiation and coalescence of neural crest--derived cells into myenteric plexuses in explants was repressed by the addition of Shh. Our data suggest that Shh controls the proliferation and differentiation of NCCs and modulates the responsiveness of NCCs toward GDNF inductions.

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Shh represses the formation of neural plexuses. Sections of X-gal/IPTG-stained b3-IIIa-LacZ explants that had been treated with (Shh) or without (control) Shh for 2 d were stained for SMA (A, B, E, and F; brown), and NCCs were localized by X-gal/IPTG staining (blue). Sections of Shh-treated or control nontransgenic explants were stained for TUJ1 (C, D, G, and H; brown). Small clumps of NCCs (stained blue) were localized to the explant surface (E, open arrowheads). The boxed regions were magnified as shown on the right. Gut explants were precultured for 2 d with or without Shh, and the medium was replaced by medium with or without GDNF and incubated for two more days. The cultures were stained for TUJ1 (I, red). Numbers of migratory cells on filters in different treatments were quantified by flow cytometry. Average cell count ± SEM were determined and shown as a histogram (n, number of explants in each treatment). Sections of Shh-treated transgenic explant were stained with anti-Shh antibody (J), blocking solution (M), or isotype control antibody (N). Immunofluorescence (K) and phase-contrast views (L) of the control explant that was stained with anti-Shh antibody are shown. (J) Serosa (arrowheads) of Shh-treated explants displayed strong immunoreactivity for Shh. The β-galactosidase–expressing NCCs (L, asterisks) at the presumptive myenteric region of control explants showed strong auto-fluorescence (K, asterisks). Photos C, D, G, and H were taken at the same magnification. Photos J–N were taken at the same magnification. m, mucosa. Bars: (A and E and J–N) 50 μm; (B and F) 25 μm; (C, D, and G–I) 100 μm.
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fig7: Shh represses the formation of neural plexuses. Sections of X-gal/IPTG-stained b3-IIIa-LacZ explants that had been treated with (Shh) or without (control) Shh for 2 d were stained for SMA (A, B, E, and F; brown), and NCCs were localized by X-gal/IPTG staining (blue). Sections of Shh-treated or control nontransgenic explants were stained for TUJ1 (C, D, G, and H; brown). Small clumps of NCCs (stained blue) were localized to the explant surface (E, open arrowheads). The boxed regions were magnified as shown on the right. Gut explants were precultured for 2 d with or without Shh, and the medium was replaced by medium with or without GDNF and incubated for two more days. The cultures were stained for TUJ1 (I, red). Numbers of migratory cells on filters in different treatments were quantified by flow cytometry. Average cell count ± SEM were determined and shown as a histogram (n, number of explants in each treatment). Sections of Shh-treated transgenic explant were stained with anti-Shh antibody (J), blocking solution (M), or isotype control antibody (N). Immunofluorescence (K) and phase-contrast views (L) of the control explant that was stained with anti-Shh antibody are shown. (J) Serosa (arrowheads) of Shh-treated explants displayed strong immunoreactivity for Shh. The β-galactosidase–expressing NCCs (L, asterisks) at the presumptive myenteric region of control explants showed strong auto-fluorescence (K, asterisks). Photos C, D, G, and H were taken at the same magnification. Photos J–N were taken at the same magnification. m, mucosa. Bars: (A and E and J–N) 50 μm; (B and F) 25 μm; (C, D, and G–I) 100 μm.

Mentions: Sections of Shh-treated explants and control explants (without Shh) of b3-IIIa-LacZ mice were stained for SMA and TUJ1 to study the concentric organization of gut explant and neuronal differentiation of NCCs. Two distinct layers of SMA+ myofibroblasts were detected in the mesenchyme of control explant suggesting that the circular and longitudinal muscle were developing properly (Fig. 7, A and B). NCCs (Fig. 7, A and B, blue) and TUJ1+ neurons (Fig. 7, C and D) coalesced at the presumptive myenteric region between the developing muscle layers. In contrast, well-defined layers of SMA+ myofibroblasts were not developed in Shh-treated explants, and the presumptive myenteric region was indiscernible (Fig. 7, E and F). In Shh-treated explants, NCCs randomly aggregated into clumps (Fig. 7 E, arrowheads), and TUJ1+ neurons were sparse and randomly distributed (Fig. 7, G and H). These findings indicated that neuronal differentiation of NCCs was repressed by Shh, and that neuronal cells failed to coalesce at the presumptive myenteric region. To test if these undifferentiated NCCs of Shh- treated explants could migrate in response to GDNF, we pretreated explants with or without Shh for 2 d, then replaced the medium with GDNF containing medium, and cultured for another 2 d. Shh pretreatment increased the number of migratory cells by twofold (P = 0.035, t test) as compared with those pretreated with the control medium (Fig. 7 I). Distributions of Shh in Shh-treated and control explants of b3-IIIa-LacZ mice were analyzed by immunofluorescence. The Shh immunofluorescence signal in control explant (Fig. 7 K) was strong at the mucosa, whereas the signal at the mesenchyme declined the further it was away from the mucosa, which was similar to that of mouse embryonic gut (compare Fig. 7 K with Fig. 1 L). The fluorescence signal at the gut mesenchyme (Fig. 7, asterisk) of the control explant was the auto-fluorescence of β-galactosidase–expressing NCCs (Fig. 7, blue) at the presumptive myenteric region (compare Fig. 7 K and Fig. 7 L). A strong Shh immunofluorescence signal was detected across the gut radius at the mucosa, mesenchyme, and serosa in Shh-treated explants (Fig. 7 J, arrowheads). This finding indicated that Shh treatment disrupted the endogenous Shh gradient across the gut radius in which Shh concentration declined from the mucosa toward the serosa. Staining without 5E1 or with mouse IgG isotype control antibody gave no signal (Fig. 7, M and N).


Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut.

Fu M, Lui VC, Sham MH, Pachnis V, Tam PK - J. Cell Biol. (2004)

Shh represses the formation of neural plexuses. Sections of X-gal/IPTG-stained b3-IIIa-LacZ explants that had been treated with (Shh) or without (control) Shh for 2 d were stained for SMA (A, B, E, and F; brown), and NCCs were localized by X-gal/IPTG staining (blue). Sections of Shh-treated or control nontransgenic explants were stained for TUJ1 (C, D, G, and H; brown). Small clumps of NCCs (stained blue) were localized to the explant surface (E, open arrowheads). The boxed regions were magnified as shown on the right. Gut explants were precultured for 2 d with or without Shh, and the medium was replaced by medium with or without GDNF and incubated for two more days. The cultures were stained for TUJ1 (I, red). Numbers of migratory cells on filters in different treatments were quantified by flow cytometry. Average cell count ± SEM were determined and shown as a histogram (n, number of explants in each treatment). Sections of Shh-treated transgenic explant were stained with anti-Shh antibody (J), blocking solution (M), or isotype control antibody (N). Immunofluorescence (K) and phase-contrast views (L) of the control explant that was stained with anti-Shh antibody are shown. (J) Serosa (arrowheads) of Shh-treated explants displayed strong immunoreactivity for Shh. The β-galactosidase–expressing NCCs (L, asterisks) at the presumptive myenteric region of control explants showed strong auto-fluorescence (K, asterisks). Photos C, D, G, and H were taken at the same magnification. Photos J–N were taken at the same magnification. m, mucosa. Bars: (A and E and J–N) 50 μm; (B and F) 25 μm; (C, D, and G–I) 100 μm.
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fig7: Shh represses the formation of neural plexuses. Sections of X-gal/IPTG-stained b3-IIIa-LacZ explants that had been treated with (Shh) or without (control) Shh for 2 d were stained for SMA (A, B, E, and F; brown), and NCCs were localized by X-gal/IPTG staining (blue). Sections of Shh-treated or control nontransgenic explants were stained for TUJ1 (C, D, G, and H; brown). Small clumps of NCCs (stained blue) were localized to the explant surface (E, open arrowheads). The boxed regions were magnified as shown on the right. Gut explants were precultured for 2 d with or without Shh, and the medium was replaced by medium with or without GDNF and incubated for two more days. The cultures were stained for TUJ1 (I, red). Numbers of migratory cells on filters in different treatments were quantified by flow cytometry. Average cell count ± SEM were determined and shown as a histogram (n, number of explants in each treatment). Sections of Shh-treated transgenic explant were stained with anti-Shh antibody (J), blocking solution (M), or isotype control antibody (N). Immunofluorescence (K) and phase-contrast views (L) of the control explant that was stained with anti-Shh antibody are shown. (J) Serosa (arrowheads) of Shh-treated explants displayed strong immunoreactivity for Shh. The β-galactosidase–expressing NCCs (L, asterisks) at the presumptive myenteric region of control explants showed strong auto-fluorescence (K, asterisks). Photos C, D, G, and H were taken at the same magnification. Photos J–N were taken at the same magnification. m, mucosa. Bars: (A and E and J–N) 50 μm; (B and F) 25 μm; (C, D, and G–I) 100 μm.
Mentions: Sections of Shh-treated explants and control explants (without Shh) of b3-IIIa-LacZ mice were stained for SMA and TUJ1 to study the concentric organization of gut explant and neuronal differentiation of NCCs. Two distinct layers of SMA+ myofibroblasts were detected in the mesenchyme of control explant suggesting that the circular and longitudinal muscle were developing properly (Fig. 7, A and B). NCCs (Fig. 7, A and B, blue) and TUJ1+ neurons (Fig. 7, C and D) coalesced at the presumptive myenteric region between the developing muscle layers. In contrast, well-defined layers of SMA+ myofibroblasts were not developed in Shh-treated explants, and the presumptive myenteric region was indiscernible (Fig. 7, E and F). In Shh-treated explants, NCCs randomly aggregated into clumps (Fig. 7 E, arrowheads), and TUJ1+ neurons were sparse and randomly distributed (Fig. 7, G and H). These findings indicated that neuronal differentiation of NCCs was repressed by Shh, and that neuronal cells failed to coalesce at the presumptive myenteric region. To test if these undifferentiated NCCs of Shh- treated explants could migrate in response to GDNF, we pretreated explants with or without Shh for 2 d, then replaced the medium with GDNF containing medium, and cultured for another 2 d. Shh pretreatment increased the number of migratory cells by twofold (P = 0.035, t test) as compared with those pretreated with the control medium (Fig. 7 I). Distributions of Shh in Shh-treated and control explants of b3-IIIa-LacZ mice were analyzed by immunofluorescence. The Shh immunofluorescence signal in control explant (Fig. 7 K) was strong at the mucosa, whereas the signal at the mesenchyme declined the further it was away from the mucosa, which was similar to that of mouse embryonic gut (compare Fig. 7 K with Fig. 1 L). The fluorescence signal at the gut mesenchyme (Fig. 7, asterisk) of the control explant was the auto-fluorescence of β-galactosidase–expressing NCCs (Fig. 7, blue) at the presumptive myenteric region (compare Fig. 7 K and Fig. 7 L). A strong Shh immunofluorescence signal was detected across the gut radius at the mucosa, mesenchyme, and serosa in Shh-treated explants (Fig. 7 J, arrowheads). This finding indicated that Shh treatment disrupted the endogenous Shh gradient across the gut radius in which Shh concentration declined from the mucosa toward the serosa. Staining without 5E1 or with mouse IgG isotype control antibody gave no signal (Fig. 7, M and N).

Bottom Line: The pro-neurogenic effect of glial cell line--derived neurotrophic factor (GDNF) on NCCs was abolished by Shh.In gut explants, NCCs migrated from the explants onto the adjacent substratum if GDNF was added, whereas addition of Shh abolished this migration.Our data suggest that Shh controls the proliferation and differentiation of NCCs and modulates the responsiveness of NCCs toward GDNF inductions.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, The University of Hong Kong, 21 Sassoon Rd., Pokfulam, Hong Kong, HKSAR China.

ABSTRACT
Enteric neural crest cells (NCCs) migrate and colonize the entire gut and proliferate and differentiate into neurons and glia of the enteric nervous system in vertebrate embryos. We have investigated the mitogenic and morphogenic functions of Sonic hedgehog (Shh) on enteric NCCs in cell and organ culture. Enteric NCCs expressed Shh receptor Patched and transcripts encoding the Shh signal transducer (Gli1). Shh promoted the proliferation and inhibited the differentiation of NCCs. The pro-neurogenic effect of glial cell line--derived neurotrophic factor (GDNF) on NCCs was abolished by Shh. In gut explants, NCCs migrated from the explants onto the adjacent substratum if GDNF was added, whereas addition of Shh abolished this migration. Neuronal differentiation and coalescence of neural crest--derived cells into myenteric plexuses in explants was repressed by the addition of Shh. Our data suggest that Shh controls the proliferation and differentiation of NCCs and modulates the responsiveness of NCCs toward GDNF inductions.

Show MeSH
Related in: MedlinePlus