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Wnt activity guides facial branchiomotor neuron migration, and involves the PCP pathway and JNK and ROCK kinases.

Vivancos V, Chen P, Spassky N, Qian D, Dabdoub A, Kelley M, Studer M, Guthrie S - Neural Dev (2009)

Bottom Line: We found that Wnt5a is expressed in a caudal high to rostral low gradient in the hindbrain.Inhibition of JNK and ROCK kinases strongly and specifically reduced the FBM migration, as well as blocked the chemoattractant effects of ectopic Wnt proteins.These results provide in vivo evidence that Wnts chemoattract mammalian FBM neurons and that Wnt5a is a candidate to mediate this process.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Developmental Neurobiology, King's College, Guy's Campus, London, SE1 1UL, UK. valerie.vivancos@kcl.ac.uk

ABSTRACT

Background: Wnt proteins play roles in many biological processes, including axon guidance and cell migration. In the mammalian hindbrain, facial branchiomotor (FBM) neurons undergo a striking rostral to caudal migration, yet little is known of the underlying molecular mechanisms. In this study, we investigated a possible role of Wnts and the planar cell polarity (PCP) pathway in this process.

Results: Here we demonstrate a novel role for Wnt proteins in guiding FBM neurons during their rostral to caudal migration in the hindbrain. We found that Wnt5a is expressed in a caudal high to rostral low gradient in the hindbrain. Wnt-coated beads chemoattracted FBM neurons to ectopic positions in an explant migration assay. The rostrocaudal FBM migration was moderately perturbed in Wnt5a mutant embryos and severely disrupted in Frizzled3 mutant mouse embryos, and was aberrant following inhibition of Wnt function by secreted Frizzled-related proteins. We also show the involvement of the Wnt/PCP pathway in mammalian FBM neuron migration. Thus, mutations in two PCP genes, Vangl2 and Scribble, caused severe defects in FBM migration. Inhibition of JNK and ROCK kinases strongly and specifically reduced the FBM migration, as well as blocked the chemoattractant effects of ectopic Wnt proteins.

Conclusion: These results provide in vivo evidence that Wnts chemoattract mammalian FBM neurons and that Wnt5a is a candidate to mediate this process. Molecules of the PCP pathway and the JNK and ROCK kinases also play a role in the FBM migration and are likely mediators of Wnt signalling.

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Chemoattractant effect of Wnt proteins in hindbrain explants. (A-B') Embryonic stage (E)11.5 mouse hindbrain explants with Wnt-coated beads placed in a region of rhombomere (r)4 rostral and lateral to the facial branchiomotor migratory stream: (A) phosphate-buffered saline (PBS) control; (B) Wnt protein; (A', B') higher magnifications of regions in boxes in (A, B), respectively. White circles mark beads. Scale bars: 250 μm in (A, B); 125 μm in (A', B'). (C, D) Quantification of bead experiments, with proteins as indicated. (C) Wnt7a versus control p < 0.001, Wnt5a versus control p < 0.001 (indicated by asterisks), n = 25 explants in each group. (D) Vascular endothelial growth factor (VEGF) versus control p < 0.01 (positive control; indicated by single asterisk). Semaphorin 3A (Sema3A) versus control p > 0.05 (negative control; indicated by double asterisks), n = 10 explants in each group.
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Figure 2: Chemoattractant effect of Wnt proteins in hindbrain explants. (A-B') Embryonic stage (E)11.5 mouse hindbrain explants with Wnt-coated beads placed in a region of rhombomere (r)4 rostral and lateral to the facial branchiomotor migratory stream: (A) phosphate-buffered saline (PBS) control; (B) Wnt protein; (A', B') higher magnifications of regions in boxes in (A, B), respectively. White circles mark beads. Scale bars: 250 μm in (A, B); 125 μm in (A', B'). (C, D) Quantification of bead experiments, with proteins as indicated. (C) Wnt7a versus control p < 0.001, Wnt5a versus control p < 0.001 (indicated by asterisks), n = 25 explants in each group. (D) Vascular endothelial growth factor (VEGF) versus control p < 0.01 (positive control; indicated by single asterisk). Semaphorin 3A (Sema3A) versus control p > 0.05 (negative control; indicated by double asterisks), n = 10 explants in each group.

Mentions: We tested the effects of Wnt proteins on this migration pattern, using beads soaked in Wnt5a or Wnt7a protein (or PBS controls) in E11.5 hindbrain explants cultured for 48 hours. These are 'non-canonical' Wnts, which have been linked to convergent extension movements in fish and frogs (reviewed by [5,31]). When PBS-soaked control beads were placed unilaterally in rostral r4, we found that the FBM migration resembled that in controls, that is, cells were not deflected from their normal migration route (Figure 2A, A'). However, placement of beads soaked in Wnt5a or Wnt7a protein led to a coalescence of FBM neurons around the beads, suggesting that there was a chemoattractant effect (Figure 2B, B'). FBM neurons in r4 and also in r5 migrated laterally, and in some cases cells from the contralateral side also moved across the midline towards the beads. Three-dimensional confocal images of these explants suggested that FBM neurons had collected around the beads. Explants containing either PBS-soaked or Wnt-soaked beads were then scored blind as to whether FBM neurons were deflected from their normal course ('attraction') or not ('no attraction'). Quantification and statistical analysis showed that this effect was significantly different from controls (Figure 2C). We also quantified migration in the presence of Wnt-coated and PBS control beads by pixel counting using the Scion image programme (see Materials and methods; Additional file 1A, B). This method also showed a significant difference between the two groups. In separate experiments, Wnt beads were also presented in r6/r7 to test whether they could attract FBM neurons caudally. An effect was detected, but showed somewhat less clearly that FBM neurons deviated from their pathway than that obtained from rostral placement of beads, possibly because of the proximity to the normal migration route/FBM nucleus (data not shown). However, our data are consistent with a role for Wnts in the caudal and lateral displacement of FBM neurons.


Wnt activity guides facial branchiomotor neuron migration, and involves the PCP pathway and JNK and ROCK kinases.

Vivancos V, Chen P, Spassky N, Qian D, Dabdoub A, Kelley M, Studer M, Guthrie S - Neural Dev (2009)

Chemoattractant effect of Wnt proteins in hindbrain explants. (A-B') Embryonic stage (E)11.5 mouse hindbrain explants with Wnt-coated beads placed in a region of rhombomere (r)4 rostral and lateral to the facial branchiomotor migratory stream: (A) phosphate-buffered saline (PBS) control; (B) Wnt protein; (A', B') higher magnifications of regions in boxes in (A, B), respectively. White circles mark beads. Scale bars: 250 μm in (A, B); 125 μm in (A', B'). (C, D) Quantification of bead experiments, with proteins as indicated. (C) Wnt7a versus control p < 0.001, Wnt5a versus control p < 0.001 (indicated by asterisks), n = 25 explants in each group. (D) Vascular endothelial growth factor (VEGF) versus control p < 0.01 (positive control; indicated by single asterisk). Semaphorin 3A (Sema3A) versus control p > 0.05 (negative control; indicated by double asterisks), n = 10 explants in each group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2654884&req=5

Figure 2: Chemoattractant effect of Wnt proteins in hindbrain explants. (A-B') Embryonic stage (E)11.5 mouse hindbrain explants with Wnt-coated beads placed in a region of rhombomere (r)4 rostral and lateral to the facial branchiomotor migratory stream: (A) phosphate-buffered saline (PBS) control; (B) Wnt protein; (A', B') higher magnifications of regions in boxes in (A, B), respectively. White circles mark beads. Scale bars: 250 μm in (A, B); 125 μm in (A', B'). (C, D) Quantification of bead experiments, with proteins as indicated. (C) Wnt7a versus control p < 0.001, Wnt5a versus control p < 0.001 (indicated by asterisks), n = 25 explants in each group. (D) Vascular endothelial growth factor (VEGF) versus control p < 0.01 (positive control; indicated by single asterisk). Semaphorin 3A (Sema3A) versus control p > 0.05 (negative control; indicated by double asterisks), n = 10 explants in each group.
Mentions: We tested the effects of Wnt proteins on this migration pattern, using beads soaked in Wnt5a or Wnt7a protein (or PBS controls) in E11.5 hindbrain explants cultured for 48 hours. These are 'non-canonical' Wnts, which have been linked to convergent extension movements in fish and frogs (reviewed by [5,31]). When PBS-soaked control beads were placed unilaterally in rostral r4, we found that the FBM migration resembled that in controls, that is, cells were not deflected from their normal migration route (Figure 2A, A'). However, placement of beads soaked in Wnt5a or Wnt7a protein led to a coalescence of FBM neurons around the beads, suggesting that there was a chemoattractant effect (Figure 2B, B'). FBM neurons in r4 and also in r5 migrated laterally, and in some cases cells from the contralateral side also moved across the midline towards the beads. Three-dimensional confocal images of these explants suggested that FBM neurons had collected around the beads. Explants containing either PBS-soaked or Wnt-soaked beads were then scored blind as to whether FBM neurons were deflected from their normal course ('attraction') or not ('no attraction'). Quantification and statistical analysis showed that this effect was significantly different from controls (Figure 2C). We also quantified migration in the presence of Wnt-coated and PBS control beads by pixel counting using the Scion image programme (see Materials and methods; Additional file 1A, B). This method also showed a significant difference between the two groups. In separate experiments, Wnt beads were also presented in r6/r7 to test whether they could attract FBM neurons caudally. An effect was detected, but showed somewhat less clearly that FBM neurons deviated from their pathway than that obtained from rostral placement of beads, possibly because of the proximity to the normal migration route/FBM nucleus (data not shown). However, our data are consistent with a role for Wnts in the caudal and lateral displacement of FBM neurons.

Bottom Line: We found that Wnt5a is expressed in a caudal high to rostral low gradient in the hindbrain.Inhibition of JNK and ROCK kinases strongly and specifically reduced the FBM migration, as well as blocked the chemoattractant effects of ectopic Wnt proteins.These results provide in vivo evidence that Wnts chemoattract mammalian FBM neurons and that Wnt5a is a candidate to mediate this process.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Developmental Neurobiology, King's College, Guy's Campus, London, SE1 1UL, UK. valerie.vivancos@kcl.ac.uk

ABSTRACT

Background: Wnt proteins play roles in many biological processes, including axon guidance and cell migration. In the mammalian hindbrain, facial branchiomotor (FBM) neurons undergo a striking rostral to caudal migration, yet little is known of the underlying molecular mechanisms. In this study, we investigated a possible role of Wnts and the planar cell polarity (PCP) pathway in this process.

Results: Here we demonstrate a novel role for Wnt proteins in guiding FBM neurons during their rostral to caudal migration in the hindbrain. We found that Wnt5a is expressed in a caudal high to rostral low gradient in the hindbrain. Wnt-coated beads chemoattracted FBM neurons to ectopic positions in an explant migration assay. The rostrocaudal FBM migration was moderately perturbed in Wnt5a mutant embryos and severely disrupted in Frizzled3 mutant mouse embryos, and was aberrant following inhibition of Wnt function by secreted Frizzled-related proteins. We also show the involvement of the Wnt/PCP pathway in mammalian FBM neuron migration. Thus, mutations in two PCP genes, Vangl2 and Scribble, caused severe defects in FBM migration. Inhibition of JNK and ROCK kinases strongly and specifically reduced the FBM migration, as well as blocked the chemoattractant effects of ectopic Wnt proteins.

Conclusion: These results provide in vivo evidence that Wnts chemoattract mammalian FBM neurons and that Wnt5a is a candidate to mediate this process. Molecules of the PCP pathway and the JNK and ROCK kinases also play a role in the FBM migration and are likely mediators of Wnt signalling.

Show MeSH
Related in: MedlinePlus