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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 promotes the proliferation of NCCs. (A) Cytopsin preparations of neurospheres that had been cultured without (control) or with (Shh) Shh were stained for TUJ1 (green) or Ret (red). TUNEL was performed to localize apoptotic cells (green; arrowhead) in neurospheres cultured without (control; B and C) or with Shh (D and E). Control neurosphere (10 d culture) contained very few BrdU+ cells (F). In contrast, neurospheres cultured with Shh for 10 d contained numerous BrdU+ cells (G). Bar, 50 μm.
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fig3: Shh promotes the proliferation of NCCs. (A) Cytopsin preparations of neurospheres that had been cultured without (control) or with (Shh) Shh were stained for TUJ1 (green) or Ret (red). TUNEL was performed to localize apoptotic cells (green; arrowhead) in neurospheres cultured without (control; B and C) or with Shh (D and E). Control neurosphere (10 d culture) contained very few BrdU+ cells (F). In contrast, neurospheres cultured with Shh for 10 d contained numerous BrdU+ cells (G). Bar, 50 μm.

Mentions: To study the effect of Shh on the growth and differentiation of enteric NCCs, we cultured neurospheres in the presence or absence of Shh and analyzed the cultures by immunostaining using anti-Ret and anti-TUJ1 antibodies (Fig. 3 A). NCCs maintained Ret expression and expressed neuron markers such as TUJ1, as they were committed to neuronal lineage. In contrast, NCCs switched off expressing Ret once they were committed to glial differentiation. We described NCCs as Ret+/TUJ1− and neuron progenitors/neurons as Ret+/TUJ1+ cells in our cultures. Numbers of cells expressing Ret with or without coexpression of TUJ1 in control and Shh-treated neurospheres were counted (Table II). NCCs (Ret+/TUJ1−) were more abundant in Shh culture than in the control medium. Conversely, neuron progenitors/neurons (Ret+/TUJ1+) were less abundant in culture with Shh than in the control medium. TUNEL staining on cytospin preparations of day 10 neurospheres revealed that apoptotic cells were rarely seen in cultures with or without Shh (Fig. 3, B–E). Shh-treated neurospheres (Fig. 3 G) contained 3.5-fold more BrdU+ cells than that in control (without Shh; Fig. 3 F; BrdU+ cells in Shh treatment vs. control: 74 ± 2.7 vs. 20 ± 1.4; P = 0.0001; t test; n = 10).


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 promotes the proliferation of NCCs. (A) Cytopsin preparations of neurospheres that had been cultured without (control) or with (Shh) Shh were stained for TUJ1 (green) or Ret (red). TUNEL was performed to localize apoptotic cells (green; arrowhead) in neurospheres cultured without (control; B and C) or with Shh (D and E). Control neurosphere (10 d culture) contained very few BrdU+ cells (F). In contrast, neurospheres cultured with Shh for 10 d contained numerous BrdU+ cells (G). Bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172437&req=5

fig3: Shh promotes the proliferation of NCCs. (A) Cytopsin preparations of neurospheres that had been cultured without (control) or with (Shh) Shh were stained for TUJ1 (green) or Ret (red). TUNEL was performed to localize apoptotic cells (green; arrowhead) in neurospheres cultured without (control; B and C) or with Shh (D and E). Control neurosphere (10 d culture) contained very few BrdU+ cells (F). In contrast, neurospheres cultured with Shh for 10 d contained numerous BrdU+ cells (G). Bar, 50 μm.
Mentions: To study the effect of Shh on the growth and differentiation of enteric NCCs, we cultured neurospheres in the presence or absence of Shh and analyzed the cultures by immunostaining using anti-Ret and anti-TUJ1 antibodies (Fig. 3 A). NCCs maintained Ret expression and expressed neuron markers such as TUJ1, as they were committed to neuronal lineage. In contrast, NCCs switched off expressing Ret once they were committed to glial differentiation. We described NCCs as Ret+/TUJ1− and neuron progenitors/neurons as Ret+/TUJ1+ cells in our cultures. Numbers of cells expressing Ret with or without coexpression of TUJ1 in control and Shh-treated neurospheres were counted (Table II). NCCs (Ret+/TUJ1−) were more abundant in Shh culture than in the control medium. Conversely, neuron progenitors/neurons (Ret+/TUJ1+) were less abundant in culture with Shh than in the control medium. TUNEL staining on cytospin preparations of day 10 neurospheres revealed that apoptotic cells were rarely seen in cultures with or without Shh (Fig. 3, B–E). Shh-treated neurospheres (Fig. 3 G) contained 3.5-fold more BrdU+ cells than that in control (without Shh; Fig. 3 F; BrdU+ cells in Shh treatment vs. control: 74 ± 2.7 vs. 20 ± 1.4; P = 0.0001; t test; n = 10).

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