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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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Inhibition of facial branchiomotor (FBM) migration and of Wnt-mediated attraction. (A, D, G) Control explants cultured for 48 hours and immunostained with anti-Islet 1/2 antibody with FBM neurons located in their final position forming the facial motor nuclei in rhombomere (r)6. (B, E, H) Explants treated with c-Jun amino-terminal kinase (JNK) inhibitor (SP600125), Rho kinase (ROCK) inhibitor (Y27632) and myosin light chain kinase inhibitor (ML-7) as labelled. Scale bars: 250 μm (A, B, D, E, G, H). (C, F, I) Quantification of the effects of inhibitors on FBM migration in explants. The x-axis is the migration scale where 1 = loss of migration, 2 = intermediate loss of migration, 3 = normal migration. The y-axis is the percentage of explants in a particular category. Inhibitor-treated versus control p < 0.001 (indicated by asterisk). (J, K) Quantification of the effect of inhibitors on Wnt5A attractive beads. The attractive effect of Wnt5A ectopic beads is inhibited by adding inhibitors SP600125 (J) and Y27632 (K). Inhibitor-treated versus control p > 0.05 (indicated by asterisk). (C, F, I, J, K) N = 15–20 explants in each group.
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Figure 6: Inhibition of facial branchiomotor (FBM) migration and of Wnt-mediated attraction. (A, D, G) Control explants cultured for 48 hours and immunostained with anti-Islet 1/2 antibody with FBM neurons located in their final position forming the facial motor nuclei in rhombomere (r)6. (B, E, H) Explants treated with c-Jun amino-terminal kinase (JNK) inhibitor (SP600125), Rho kinase (ROCK) inhibitor (Y27632) and myosin light chain kinase inhibitor (ML-7) as labelled. Scale bars: 250 μm (A, B, D, E, G, H). (C, F, I) Quantification of the effects of inhibitors on FBM migration in explants. The x-axis is the migration scale where 1 = loss of migration, 2 = intermediate loss of migration, 3 = normal migration. The y-axis is the percentage of explants in a particular category. Inhibitor-treated versus control p < 0.001 (indicated by asterisk). (J, K) Quantification of the effect of inhibitors on Wnt5A attractive beads. The attractive effect of Wnt5A ectopic beads is inhibited by adding inhibitors SP600125 (J) and Y27632 (K). Inhibitor-treated versus control p > 0.05 (indicated by asterisk). (C, F, I, J, K) N = 15–20 explants in each group.

Mentions: In order to dissect the signalling pathways involved in FBM migration, we applied pharmacological inhibitors of candidate molecules to the FBM migration assay. Inhibitors were applied at the beginning of the culture period and the distribution of Islet-1/2-positive FBM neurons was compared between inhibitor-treated explants and those treated with vehicle alone (DMSO controls). Migration was scored on a scale of 1–3 as for SFRP-treated explants. Explants treated with JNK inhibitor (SP600125) showed a dose-dependent disruption of migration, with 5 μM resulting in a moderate inhibition (57.2% of explants were grade 1 (loss of migration); data not shown) and 10 μM producing an effect in which FBM neurons stopped in r5 (68% of explants were scored as grade 1; Figure 6A–C). Inhibitors of ROCK (Y27632) or its downstream target MLCK (ML-7) both caused FBM migration defects, but in fewer cases, with 40–50% of explants showing grade 1 morphology (Figure 6D–I).


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)

Inhibition of facial branchiomotor (FBM) migration and of Wnt-mediated attraction. (A, D, G) Control explants cultured for 48 hours and immunostained with anti-Islet 1/2 antibody with FBM neurons located in their final position forming the facial motor nuclei in rhombomere (r)6. (B, E, H) Explants treated with c-Jun amino-terminal kinase (JNK) inhibitor (SP600125), Rho kinase (ROCK) inhibitor (Y27632) and myosin light chain kinase inhibitor (ML-7) as labelled. Scale bars: 250 μm (A, B, D, E, G, H). (C, F, I) Quantification of the effects of inhibitors on FBM migration in explants. The x-axis is the migration scale where 1 = loss of migration, 2 = intermediate loss of migration, 3 = normal migration. The y-axis is the percentage of explants in a particular category. Inhibitor-treated versus control p < 0.001 (indicated by asterisk). (J, K) Quantification of the effect of inhibitors on Wnt5A attractive beads. The attractive effect of Wnt5A ectopic beads is inhibited by adding inhibitors SP600125 (J) and Y27632 (K). Inhibitor-treated versus control p > 0.05 (indicated by asterisk). (C, F, I, J, K) N = 15–20 explants in each group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Inhibition of facial branchiomotor (FBM) migration and of Wnt-mediated attraction. (A, D, G) Control explants cultured for 48 hours and immunostained with anti-Islet 1/2 antibody with FBM neurons located in their final position forming the facial motor nuclei in rhombomere (r)6. (B, E, H) Explants treated with c-Jun amino-terminal kinase (JNK) inhibitor (SP600125), Rho kinase (ROCK) inhibitor (Y27632) and myosin light chain kinase inhibitor (ML-7) as labelled. Scale bars: 250 μm (A, B, D, E, G, H). (C, F, I) Quantification of the effects of inhibitors on FBM migration in explants. The x-axis is the migration scale where 1 = loss of migration, 2 = intermediate loss of migration, 3 = normal migration. The y-axis is the percentage of explants in a particular category. Inhibitor-treated versus control p < 0.001 (indicated by asterisk). (J, K) Quantification of the effect of inhibitors on Wnt5A attractive beads. The attractive effect of Wnt5A ectopic beads is inhibited by adding inhibitors SP600125 (J) and Y27632 (K). Inhibitor-treated versus control p > 0.05 (indicated by asterisk). (C, F, I, J, K) N = 15–20 explants in each group.
Mentions: In order to dissect the signalling pathways involved in FBM migration, we applied pharmacological inhibitors of candidate molecules to the FBM migration assay. Inhibitors were applied at the beginning of the culture period and the distribution of Islet-1/2-positive FBM neurons was compared between inhibitor-treated explants and those treated with vehicle alone (DMSO controls). Migration was scored on a scale of 1–3 as for SFRP-treated explants. Explants treated with JNK inhibitor (SP600125) showed a dose-dependent disruption of migration, with 5 μM resulting in a moderate inhibition (57.2% of explants were grade 1 (loss of migration); data not shown) and 10 μM producing an effect in which FBM neurons stopped in r5 (68% of explants were scored as grade 1; Figure 6A–C). Inhibitors of ROCK (Y27632) or its downstream target MLCK (ML-7) both caused FBM migration defects, but in fewer cases, with 40–50% of explants showing grade 1 morphology (Figure 6D–I).

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