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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 restricts the migration of NCCs. X-gal/IPTG staining of E11.5 b3-IIIa-LacZ transgenic embryo indicated β-galactosidase activities (blue) were detected at the anterior spinal cord, hindbrain, and intestine (A and B, insets) reaching the cecum (C, arrowhead). Sections of the X-gal/IPTG-stained E11.5 foregut revealed β-galactosidase–expressing cells in the mesenchyme (D) and colocalized with p75NTR immunostaining signals (E). Gut explants of E11.5 transgenic mouse embryos were established as shown (F). After 3 d of incubation with GDNF (G), GDNF + Shh (H), and GDNF + Shh + Shh neutralizing antibody (I), explants together with the filter paper were stained with X-gal/IPTG. Explants of E11.5 nontransgenic mouse guts were incubated in different conditions for 3 d and stained for Ret (J–M, red). Numbers of migratory cells on filters in different treatments were quantified by flow cytometry (N). Cells selected for counting are highlighted with circle (R1). The cells that were collected from filters for counting expressed Ret and TUJ1. Cell counts in each treatment were determined and tabulated as shown (n, number of explants in each treatment). s, stomach; c, cecum; mg, midgut; hg, hindgut. Photos J–M were taken at the same magnification. Bar, 100 μm.
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fig5: Shh restricts the migration of NCCs. X-gal/IPTG staining of E11.5 b3-IIIa-LacZ transgenic embryo indicated β-galactosidase activities (blue) were detected at the anterior spinal cord, hindbrain, and intestine (A and B, insets) reaching the cecum (C, arrowhead). Sections of the X-gal/IPTG-stained E11.5 foregut revealed β-galactosidase–expressing cells in the mesenchyme (D) and colocalized with p75NTR immunostaining signals (E). Gut explants of E11.5 transgenic mouse embryos were established as shown (F). After 3 d of incubation with GDNF (G), GDNF + Shh (H), and GDNF + Shh + Shh neutralizing antibody (I), explants together with the filter paper were stained with X-gal/IPTG. Explants of E11.5 nontransgenic mouse guts were incubated in different conditions for 3 d and stained for Ret (J–M, red). Numbers of migratory cells on filters in different treatments were quantified by flow cytometry (N). Cells selected for counting are highlighted with circle (R1). The cells that were collected from filters for counting expressed Ret and TUJ1. Cell counts in each treatment were determined and tabulated as shown (n, number of explants in each treatment). s, stomach; c, cecum; mg, midgut; hg, hindgut. Photos J–M were taken at the same magnification. Bar, 100 μm.

Mentions: To investigate the effect of Shh on NCC migration, we established ex vivo gut explant cultures of b3-IIIa-LacZ transgenic and nontransgenic mice. A 500-base cis-acting enhancer of mouse Hoxb3 gene directs the β-galactosidase expression to the hindbrain at rhombomeres 6–8, the anterior spinal cord, and the associated NCCs in b3-IIIa-LacZ transgenic mice (Yau et al., 2002). Migrating NCCs and neural plexus of transgenic gut were stained blue by X-gal/IPTG, indicative of β-galactosidase activity, and expressed NCC marker p75NTR (Fig. 5, A–E). In the presence of GDNF, β-galactosidase–expressing NCCs migrated onto the membrane (Fig. 5 G). Addition of Shh reduced NCC migration (Fig. 5 H), and Shh inhibition was partially abolished by Shh-neutralizing antibody (Fig. 5 I). Inhibition of Shh on NCC migration was also observed in nontransgenic explants (compare Fig. 5, J–M).


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 restricts the migration of NCCs. X-gal/IPTG staining of E11.5 b3-IIIa-LacZ transgenic embryo indicated β-galactosidase activities (blue) were detected at the anterior spinal cord, hindbrain, and intestine (A and B, insets) reaching the cecum (C, arrowhead). Sections of the X-gal/IPTG-stained E11.5 foregut revealed β-galactosidase–expressing cells in the mesenchyme (D) and colocalized with p75NTR immunostaining signals (E). Gut explants of E11.5 transgenic mouse embryos were established as shown (F). After 3 d of incubation with GDNF (G), GDNF + Shh (H), and GDNF + Shh + Shh neutralizing antibody (I), explants together with the filter paper were stained with X-gal/IPTG. Explants of E11.5 nontransgenic mouse guts were incubated in different conditions for 3 d and stained for Ret (J–M, red). Numbers of migratory cells on filters in different treatments were quantified by flow cytometry (N). Cells selected for counting are highlighted with circle (R1). The cells that were collected from filters for counting expressed Ret and TUJ1. Cell counts in each treatment were determined and tabulated as shown (n, number of explants in each treatment). s, stomach; c, cecum; mg, midgut; hg, hindgut. Photos J–M were taken at the same magnification. Bar, 100 μm.
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Related In: Results  -  Collection

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fig5: Shh restricts the migration of NCCs. X-gal/IPTG staining of E11.5 b3-IIIa-LacZ transgenic embryo indicated β-galactosidase activities (blue) were detected at the anterior spinal cord, hindbrain, and intestine (A and B, insets) reaching the cecum (C, arrowhead). Sections of the X-gal/IPTG-stained E11.5 foregut revealed β-galactosidase–expressing cells in the mesenchyme (D) and colocalized with p75NTR immunostaining signals (E). Gut explants of E11.5 transgenic mouse embryos were established as shown (F). After 3 d of incubation with GDNF (G), GDNF + Shh (H), and GDNF + Shh + Shh neutralizing antibody (I), explants together with the filter paper were stained with X-gal/IPTG. Explants of E11.5 nontransgenic mouse guts were incubated in different conditions for 3 d and stained for Ret (J–M, red). Numbers of migratory cells on filters in different treatments were quantified by flow cytometry (N). Cells selected for counting are highlighted with circle (R1). The cells that were collected from filters for counting expressed Ret and TUJ1. Cell counts in each treatment were determined and tabulated as shown (n, number of explants in each treatment). s, stomach; c, cecum; mg, midgut; hg, hindgut. Photos J–M were taken at the same magnification. Bar, 100 μm.
Mentions: To investigate the effect of Shh on NCC migration, we established ex vivo gut explant cultures of b3-IIIa-LacZ transgenic and nontransgenic mice. A 500-base cis-acting enhancer of mouse Hoxb3 gene directs the β-galactosidase expression to the hindbrain at rhombomeres 6–8, the anterior spinal cord, and the associated NCCs in b3-IIIa-LacZ transgenic mice (Yau et al., 2002). Migrating NCCs and neural plexus of transgenic gut were stained blue by X-gal/IPTG, indicative of β-galactosidase activity, and expressed NCC marker p75NTR (Fig. 5, A–E). In the presence of GDNF, β-galactosidase–expressing NCCs migrated onto the membrane (Fig. 5 G). Addition of Shh reduced NCC migration (Fig. 5 H), and Shh inhibition was partially abolished by Shh-neutralizing antibody (Fig. 5 I). Inhibition of Shh on NCC migration was also observed in nontransgenic explants (compare Fig. 5, J–M).

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