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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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Analysis of Vangl2 and Scribble mutant phenotypes. (A, B, D, E, G, H, I-K) In situ hybridization with Islet-1 probe on flat-mount hindbrains showing facial branchiomotor (FBM) migration in wild-type embryos, and Vangl2 and Scribble heterozygotes and homozygotes. Embryonic stages (E) and probes are as indicated. Both homozygote mutants present a very dramatic failure of FBM migration (asterisks in (D, E, G, H)) with a population of motor neurons in the floor plate (FP, arrowheads). (J, K) In heterozygous Vangl2 and Scribble (Scrb) littermates at E13.5, a stream of cells arrest in the dorsal region of rhombomere (r)5 (arrowheads) compared to wild type (WT) (C, F) Transverse sections through the hindbrains of wild-type (C) and Vangl2-/- (F) hindbrains, immunostained with anti-Islet1/2 antibody to show FBM neurons migrating laterally in r6 (C) to form the FBM nucleus (asterisk) or remaining in r4 (asterisk in F). (I). Scale bars: 250 μm in (A, B, D, E, G, H); 100 μm in (C, F); 125 μm in (I-K). TN, trigeminal motor nucleus.
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Figure 4: Analysis of Vangl2 and Scribble mutant phenotypes. (A, B, D, E, G, H, I-K) In situ hybridization with Islet-1 probe on flat-mount hindbrains showing facial branchiomotor (FBM) migration in wild-type embryos, and Vangl2 and Scribble heterozygotes and homozygotes. Embryonic stages (E) and probes are as indicated. Both homozygote mutants present a very dramatic failure of FBM migration (asterisks in (D, E, G, H)) with a population of motor neurons in the floor plate (FP, arrowheads). (J, K) In heterozygous Vangl2 and Scribble (Scrb) littermates at E13.5, a stream of cells arrest in the dorsal region of rhombomere (r)5 (arrowheads) compared to wild type (WT) (C, F) Transverse sections through the hindbrains of wild-type (C) and Vangl2-/- (F) hindbrains, immunostained with anti-Islet1/2 antibody to show FBM neurons migrating laterally in r6 (C) to form the FBM nucleus (asterisk) or remaining in r4 (asterisk in F). (I). Scale bars: 250 μm in (A, B, D, E, G, H); 100 μm in (C, F); 125 μm in (I-K). TN, trigeminal motor nucleus.

Mentions: Since it is possible that in this system Wnts operate via, or co-operate with, the PCP signalling pathway, we next analysed loop-tail and circle-tail mice, which are mutant for the Vangl2 and Scribble genes, respectively [25,33]. These mice manifest neural tube defects, including an open hindbrain. However, observation of flat-mounted preparations showed that normal rhombomere morphology was maintained. mRNA in situ hybridisation of mutant hindbrains for EphA4 and Krox-20 showed that these genes were expressed in r3 and r5, while Slit1 was expressed in the floor plate as previously reported [34-36] (Additional file 2A, B, E, F; data not shown). We therefore compared the pattern of FBM neuronal migration using in situ hybridisation for Islet-1 on wild-type, homozygous and heterozygous Vangl2 and Scribble hindbrains at E11.5 and E13.5. We observed dramatic defects in FBM migration in the homozygous embryos of both lines compared to wild-type embryos. In wild-type embryo litter-mates at E11.5, there was a normal distribution of FBM neurons, many of which had reached r5 (Figures 1 and 4A). Some had started to migrate laterally in r6 (Figure 4A, C). By contrast, in Vangl2 and Scribble mutants at E11.5, all FBM neurons had failed to migrate caudally and were still located in r4 (Figure 4D, F, G).


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)

Analysis of Vangl2 and Scribble mutant phenotypes. (A, B, D, E, G, H, I-K) In situ hybridization with Islet-1 probe on flat-mount hindbrains showing facial branchiomotor (FBM) migration in wild-type embryos, and Vangl2 and Scribble heterozygotes and homozygotes. Embryonic stages (E) and probes are as indicated. Both homozygote mutants present a very dramatic failure of FBM migration (asterisks in (D, E, G, H)) with a population of motor neurons in the floor plate (FP, arrowheads). (J, K) In heterozygous Vangl2 and Scribble (Scrb) littermates at E13.5, a stream of cells arrest in the dorsal region of rhombomere (r)5 (arrowheads) compared to wild type (WT) (C, F) Transverse sections through the hindbrains of wild-type (C) and Vangl2-/- (F) hindbrains, immunostained with anti-Islet1/2 antibody to show FBM neurons migrating laterally in r6 (C) to form the FBM nucleus (asterisk) or remaining in r4 (asterisk in F). (I). Scale bars: 250 μm in (A, B, D, E, G, H); 100 μm in (C, F); 125 μm in (I-K). TN, trigeminal motor nucleus.
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Related In: Results  -  Collection

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Figure 4: Analysis of Vangl2 and Scribble mutant phenotypes. (A, B, D, E, G, H, I-K) In situ hybridization with Islet-1 probe on flat-mount hindbrains showing facial branchiomotor (FBM) migration in wild-type embryos, and Vangl2 and Scribble heterozygotes and homozygotes. Embryonic stages (E) and probes are as indicated. Both homozygote mutants present a very dramatic failure of FBM migration (asterisks in (D, E, G, H)) with a population of motor neurons in the floor plate (FP, arrowheads). (J, K) In heterozygous Vangl2 and Scribble (Scrb) littermates at E13.5, a stream of cells arrest in the dorsal region of rhombomere (r)5 (arrowheads) compared to wild type (WT) (C, F) Transverse sections through the hindbrains of wild-type (C) and Vangl2-/- (F) hindbrains, immunostained with anti-Islet1/2 antibody to show FBM neurons migrating laterally in r6 (C) to form the FBM nucleus (asterisk) or remaining in r4 (asterisk in F). (I). Scale bars: 250 μm in (A, B, D, E, G, H); 100 μm in (C, F); 125 μm in (I-K). TN, trigeminal motor nucleus.
Mentions: Since it is possible that in this system Wnts operate via, or co-operate with, the PCP signalling pathway, we next analysed loop-tail and circle-tail mice, which are mutant for the Vangl2 and Scribble genes, respectively [25,33]. These mice manifest neural tube defects, including an open hindbrain. However, observation of flat-mounted preparations showed that normal rhombomere morphology was maintained. mRNA in situ hybridisation of mutant hindbrains for EphA4 and Krox-20 showed that these genes were expressed in r3 and r5, while Slit1 was expressed in the floor plate as previously reported [34-36] (Additional file 2A, B, E, F; data not shown). We therefore compared the pattern of FBM neuronal migration using in situ hybridisation for Islet-1 on wild-type, homozygous and heterozygous Vangl2 and Scribble hindbrains at E11.5 and E13.5. We observed dramatic defects in FBM migration in the homozygous embryos of both lines compared to wild-type embryos. In wild-type embryo litter-mates at E11.5, there was a normal distribution of FBM neurons, many of which had reached r5 (Figures 1 and 4A). Some had started to migrate laterally in r6 (Figure 4A, C). By contrast, in Vangl2 and Scribble mutants at E11.5, all FBM neurons had failed to migrate caudally and were still located in r4 (Figure 4D, F, G).

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