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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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Neurospheres contain self-renewable and multipotent NCCs. (A) Primary neurospheres were formed at day eight. After replating at a clonal density, small clusters were seen at day two (B), which domed (C) and developed into neurospheres at day five (D). Immunofluorescence analyses of cytospin preparations of neurospheres were performed to localize Ret+ cells (E and K), TUJ1+ neuron progenitors/neurons (F), TH+ neurons (G), GFAP+ glia (H), and SMA+ myofibroblasts (L). Neurosphere preparation was stained for Ret (K), SMA (L), and DAPI (M). Whole-mount immunofluorescence and BrdU incorporation assays of neurosphere showed that Ret+ cells incorporated BrdU (I and J). Immunocolocalization analyses indicated that Ptc1 (O) immunoreactivity overlapped with that of Ret (N). In contrast, cells showing intense Ptc1 staining (R, closed arrowheads) were SMA− (compare Q with R). Neurosphere preparation was stained either for Ret (N), Ptc1 (O), and DAPI (P) or for SMA (Q), Ptc1 (R), and DAPI (S). Photos A–D were taken at the same magnifications. Photos E–S were taken at the same magnifications. Bars: (A–D) 50 μm; (E–S) 25 μm.
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fig2: Neurospheres contain self-renewable and multipotent NCCs. (A) Primary neurospheres were formed at day eight. After replating at a clonal density, small clusters were seen at day two (B), which domed (C) and developed into neurospheres at day five (D). Immunofluorescence analyses of cytospin preparations of neurospheres were performed to localize Ret+ cells (E and K), TUJ1+ neuron progenitors/neurons (F), TH+ neurons (G), GFAP+ glia (H), and SMA+ myofibroblasts (L). Neurosphere preparation was stained for Ret (K), SMA (L), and DAPI (M). Whole-mount immunofluorescence and BrdU incorporation assays of neurosphere showed that Ret+ cells incorporated BrdU (I and J). Immunocolocalization analyses indicated that Ptc1 (O) immunoreactivity overlapped with that of Ret (N). In contrast, cells showing intense Ptc1 staining (R, closed arrowheads) were SMA− (compare Q with R). Neurosphere preparation was stained either for Ret (N), Ptc1 (O), and DAPI (P) or for SMA (Q), Ptc1 (R), and DAPI (S). Photos A–D were taken at the same magnifications. Photos E–S were taken at the same magnifications. Bars: (A–D) 50 μm; (E–S) 25 μm.

Mentions: To prepare the neurosphere of enteric NCCs, dissociated E11.5 mouse guts were cultured on coated dishes. Mesenchyme cells grew as a monolayer and NCCs formed clusters. Mesenchyme cells grow poorly, and were removed in each replating. Very few mesenchyme cells remained in culture after five passages (i.e., ∼8–10 d from day one of culture). Clusters of cells were first seen at 6 d. These clusters formed domes and later developed into primary neurospheres at day eight (Fig. 2 A). If neurospheres were replated at clonal density, small clusters could be observed at day two growing into secondary neurospheres at day five (Fig. 2, B–D). By replating at a clonal density, neurosphere cells were capable of being maintained in culture for longer than 60 d, which was equivalent to more than 10 passages; this indicated self-renewal of neurosphere cells.


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)

Neurospheres contain self-renewable and multipotent NCCs. (A) Primary neurospheres were formed at day eight. After replating at a clonal density, small clusters were seen at day two (B), which domed (C) and developed into neurospheres at day five (D). Immunofluorescence analyses of cytospin preparations of neurospheres were performed to localize Ret+ cells (E and K), TUJ1+ neuron progenitors/neurons (F), TH+ neurons (G), GFAP+ glia (H), and SMA+ myofibroblasts (L). Neurosphere preparation was stained for Ret (K), SMA (L), and DAPI (M). Whole-mount immunofluorescence and BrdU incorporation assays of neurosphere showed that Ret+ cells incorporated BrdU (I and J). Immunocolocalization analyses indicated that Ptc1 (O) immunoreactivity overlapped with that of Ret (N). In contrast, cells showing intense Ptc1 staining (R, closed arrowheads) were SMA− (compare Q with R). Neurosphere preparation was stained either for Ret (N), Ptc1 (O), and DAPI (P) or for SMA (Q), Ptc1 (R), and DAPI (S). Photos A–D were taken at the same magnifications. Photos E–S were taken at the same magnifications. Bars: (A–D) 50 μm; (E–S) 25 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172437&req=5

fig2: Neurospheres contain self-renewable and multipotent NCCs. (A) Primary neurospheres were formed at day eight. After replating at a clonal density, small clusters were seen at day two (B), which domed (C) and developed into neurospheres at day five (D). Immunofluorescence analyses of cytospin preparations of neurospheres were performed to localize Ret+ cells (E and K), TUJ1+ neuron progenitors/neurons (F), TH+ neurons (G), GFAP+ glia (H), and SMA+ myofibroblasts (L). Neurosphere preparation was stained for Ret (K), SMA (L), and DAPI (M). Whole-mount immunofluorescence and BrdU incorporation assays of neurosphere showed that Ret+ cells incorporated BrdU (I and J). Immunocolocalization analyses indicated that Ptc1 (O) immunoreactivity overlapped with that of Ret (N). In contrast, cells showing intense Ptc1 staining (R, closed arrowheads) were SMA− (compare Q with R). Neurosphere preparation was stained either for Ret (N), Ptc1 (O), and DAPI (P) or for SMA (Q), Ptc1 (R), and DAPI (S). Photos A–D were taken at the same magnifications. Photos E–S were taken at the same magnifications. Bars: (A–D) 50 μm; (E–S) 25 μm.
Mentions: To prepare the neurosphere of enteric NCCs, dissociated E11.5 mouse guts were cultured on coated dishes. Mesenchyme cells grew as a monolayer and NCCs formed clusters. Mesenchyme cells grow poorly, and were removed in each replating. Very few mesenchyme cells remained in culture after five passages (i.e., ∼8–10 d from day one of culture). Clusters of cells were first seen at 6 d. These clusters formed domes and later developed into primary neurospheres at day eight (Fig. 2 A). If neurospheres were replated at clonal density, small clusters could be observed at day two growing into secondary neurospheres at day five (Fig. 2, B–D). By replating at a clonal density, neurosphere cells were capable of being maintained in culture for longer than 60 d, which was equivalent to more than 10 passages; this indicated self-renewal of neurosphere cells.

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