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Dual function of Slit2 in repulsion and enhanced migration of trunk, but not vagal, neural crest cells.

De Bellard ME, Rao Y, Bronner-Fraser M - J. Cell Biol. (2003)

Bottom Line: Accordingly, only trunk neural crest cells express Robo receptors.Conversely, exposure to soluble Slit2 significantly increases the distance traversed by trunk neural crest cells.These results suggest that Slit2 can act bifunctionally, both repulsing and stimulating the motility of trunk neural crest cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
Neural crest precursors to the autonomic nervous system form different derivatives depending upon their axial level of origin; for example, vagal, but not trunk, neural crest cells form the enteric ganglia of the gut. Here, we show that Slit2 is expressed at the entrance of the gut, which is selectively invaded by vagal, but not trunk, neural crest. Accordingly, only trunk neural crest cells express Robo receptors. In vivo and in vitro experiments demonstrate that trunk, not vagal, crest cells avoid cells or cell membranes expressing Slit2, thereby contributing to the differential ability of neural crest populations to invade and innervate the gut. Conversely, exposure to soluble Slit2 significantly increases the distance traversed by trunk neural crest cells. These results suggest that Slit2 can act bifunctionally, both repulsing and stimulating the motility of trunk neural crest cells.

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Slit2 enhances trunk neural crest motility in a wound assay. Trunk neural tubes were cultured overnight on fibronectin. After one day, media was changed to one conditioned by control or Slit2-secreting cells. A wound of one to two cells width was made with a fine pipette. After 2 h, the percent of wounds with cells crossing and sealing the gap was determined. (a, unsealed) Image of a neural crest culture fixed immediately after wounding and stained with the HNK-1 antibody. (a, sealed) Image of a similar culture fixed 4 h after wounding showing that many neural crest cells have sealed the gap by this time point. (b) Primed neural crest cultures were incubated for 2 h before performing the wound with media conditioned for 5 d by control HEK cells or Slit2-secreting cells. Nonprimed corresponds to neural crest cells that were not preexposed to Slit2 in the media before the wound. Data correspond to one representative experiment out of eight. (c) Slit2 enhances wound healing of trunk, not vagal, neural crest, and this effect can be reversed by soluble Robo. The enhanced migration of trunk neural crest by Slit2 was significantly reduced by the presence of RoboN in the media. Neural crest cultures were primed for 2 h before performing the wound with media conditioned for 5 d by control HEK cells, Slit2-secreting cells, or a 1:1 combination of RoboN and Slit2 media. After 2 h of culture, the percent of wounds with cells crossing and sealing the gap was determined. Data correspond to one representative experiment of six.
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fig7: Slit2 enhances trunk neural crest motility in a wound assay. Trunk neural tubes were cultured overnight on fibronectin. After one day, media was changed to one conditioned by control or Slit2-secreting cells. A wound of one to two cells width was made with a fine pipette. After 2 h, the percent of wounds with cells crossing and sealing the gap was determined. (a, unsealed) Image of a neural crest culture fixed immediately after wounding and stained with the HNK-1 antibody. (a, sealed) Image of a similar culture fixed 4 h after wounding showing that many neural crest cells have sealed the gap by this time point. (b) Primed neural crest cultures were incubated for 2 h before performing the wound with media conditioned for 5 d by control HEK cells or Slit2-secreting cells. Nonprimed corresponds to neural crest cells that were not preexposed to Slit2 in the media before the wound. Data correspond to one representative experiment out of eight. (c) Slit2 enhances wound healing of trunk, not vagal, neural crest, and this effect can be reversed by soluble Robo. The enhanced migration of trunk neural crest by Slit2 was significantly reduced by the presence of RoboN in the media. Neural crest cultures were primed for 2 h before performing the wound with media conditioned for 5 d by control HEK cells, Slit2-secreting cells, or a 1:1 combination of RoboN and Slit2 media. After 2 h of culture, the percent of wounds with cells crossing and sealing the gap was determined. Data correspond to one representative experiment of six.

Mentions: In a second assay for Slit2's effects on migrating neural crest cells, we examined the rapidity with which neural crest cells closed an injury-induced gap within their population (Fig. 7 a). In the presence of Slit2 CM, >20% of cultures had completely sealed the gap within 2 h, compared with <10% of cultures exposed to control medium (Fig. 7 b; Table IV). Moreover, when cells were “primed” with CM before the wound assay, >40% of cultures had sealed the wound within 2 h, compared with <20% of controls (P < 0.005; Table IV). The increased migratory response occurred rapidly, within ∼1 h of exposure to Slit2. Given this short time interval, an increase in the number of migratory cells is unlikely (Mason et al., 2001). Consistent with this, no increase in cell division was observed by anti-phosphohistone3 labeling, when neural crest cells were exposed to Slit2 (unpublished data). Furthermore, we noted no alteration in cell death, as assayed by pyknotic nuclei identified by DAPI staining (unpublished data). In contrast to trunk neural crest cells, vagal neural crest cells were unaffected by the presence of Slit2 in the medium.


Dual function of Slit2 in repulsion and enhanced migration of trunk, but not vagal, neural crest cells.

De Bellard ME, Rao Y, Bronner-Fraser M - J. Cell Biol. (2003)

Slit2 enhances trunk neural crest motility in a wound assay. Trunk neural tubes were cultured overnight on fibronectin. After one day, media was changed to one conditioned by control or Slit2-secreting cells. A wound of one to two cells width was made with a fine pipette. After 2 h, the percent of wounds with cells crossing and sealing the gap was determined. (a, unsealed) Image of a neural crest culture fixed immediately after wounding and stained with the HNK-1 antibody. (a, sealed) Image of a similar culture fixed 4 h after wounding showing that many neural crest cells have sealed the gap by this time point. (b) Primed neural crest cultures were incubated for 2 h before performing the wound with media conditioned for 5 d by control HEK cells or Slit2-secreting cells. Nonprimed corresponds to neural crest cells that were not preexposed to Slit2 in the media before the wound. Data correspond to one representative experiment out of eight. (c) Slit2 enhances wound healing of trunk, not vagal, neural crest, and this effect can be reversed by soluble Robo. The enhanced migration of trunk neural crest by Slit2 was significantly reduced by the presence of RoboN in the media. Neural crest cultures were primed for 2 h before performing the wound with media conditioned for 5 d by control HEK cells, Slit2-secreting cells, or a 1:1 combination of RoboN and Slit2 media. After 2 h of culture, the percent of wounds with cells crossing and sealing the gap was determined. Data correspond to one representative experiment of six.
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fig7: Slit2 enhances trunk neural crest motility in a wound assay. Trunk neural tubes were cultured overnight on fibronectin. After one day, media was changed to one conditioned by control or Slit2-secreting cells. A wound of one to two cells width was made with a fine pipette. After 2 h, the percent of wounds with cells crossing and sealing the gap was determined. (a, unsealed) Image of a neural crest culture fixed immediately after wounding and stained with the HNK-1 antibody. (a, sealed) Image of a similar culture fixed 4 h after wounding showing that many neural crest cells have sealed the gap by this time point. (b) Primed neural crest cultures were incubated for 2 h before performing the wound with media conditioned for 5 d by control HEK cells or Slit2-secreting cells. Nonprimed corresponds to neural crest cells that were not preexposed to Slit2 in the media before the wound. Data correspond to one representative experiment out of eight. (c) Slit2 enhances wound healing of trunk, not vagal, neural crest, and this effect can be reversed by soluble Robo. The enhanced migration of trunk neural crest by Slit2 was significantly reduced by the presence of RoboN in the media. Neural crest cultures were primed for 2 h before performing the wound with media conditioned for 5 d by control HEK cells, Slit2-secreting cells, or a 1:1 combination of RoboN and Slit2 media. After 2 h of culture, the percent of wounds with cells crossing and sealing the gap was determined. Data correspond to one representative experiment of six.
Mentions: In a second assay for Slit2's effects on migrating neural crest cells, we examined the rapidity with which neural crest cells closed an injury-induced gap within their population (Fig. 7 a). In the presence of Slit2 CM, >20% of cultures had completely sealed the gap within 2 h, compared with <10% of cultures exposed to control medium (Fig. 7 b; Table IV). Moreover, when cells were “primed” with CM before the wound assay, >40% of cultures had sealed the wound within 2 h, compared with <20% of controls (P < 0.005; Table IV). The increased migratory response occurred rapidly, within ∼1 h of exposure to Slit2. Given this short time interval, an increase in the number of migratory cells is unlikely (Mason et al., 2001). Consistent with this, no increase in cell division was observed by anti-phosphohistone3 labeling, when neural crest cells were exposed to Slit2 (unpublished data). Furthermore, we noted no alteration in cell death, as assayed by pyknotic nuclei identified by DAPI staining (unpublished data). In contrast to trunk neural crest cells, vagal neural crest cells were unaffected by the presence of Slit2 in the medium.

Bottom Line: Accordingly, only trunk neural crest cells express Robo receptors.Conversely, exposure to soluble Slit2 significantly increases the distance traversed by trunk neural crest cells.These results suggest that Slit2 can act bifunctionally, both repulsing and stimulating the motility of trunk neural crest cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
Neural crest precursors to the autonomic nervous system form different derivatives depending upon their axial level of origin; for example, vagal, but not trunk, neural crest cells form the enteric ganglia of the gut. Here, we show that Slit2 is expressed at the entrance of the gut, which is selectively invaded by vagal, but not trunk, neural crest. Accordingly, only trunk neural crest cells express Robo receptors. In vivo and in vitro experiments demonstrate that trunk, not vagal, crest cells avoid cells or cell membranes expressing Slit2, thereby contributing to the differential ability of neural crest populations to invade and innervate the gut. Conversely, exposure to soluble Slit2 significantly increases the distance traversed by trunk neural crest cells. These results suggest that Slit2 can act bifunctionally, both repulsing and stimulating the motility of trunk neural crest cells.

Show MeSH
Related in: MedlinePlus