Wnt activity guides facial branchiomotor neuron migration, and involves the PCP pathway and JNK and ROCK kinases
© Vivancos et al.; licensee BioMed Central Ltd. 2009
Received: 19 June 2008
Accepted: 11 February 2009
Published: 11 February 2009
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.
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 caudalhigh to rostrallow 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.
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.
Neuronal migration is a fundamental feature of the developing nervous system . A striking migration is undertaken by the facial branchiomotor neurons (FBMs) of the mammalian embryonic hindbrain. In the mouse embryo, FBM neurons differentiate within rhombomere 4 (r4), while facial visceral motor neurons differentiate in r5 [2, 3]. FBM neurons extend axons via the facial nerve (between embryonic day (E)10.5 and E13.5); concomitantly, neuronal somata migrate caudally from r4 to r6 commencing around E10.5, and finally migrate radially within r6 to form the facial motor nucleus [2, 4]. There is very little information about the molecules that drive FBM migration in the mouse embryo. Here we demonstrate for the first time a role for the Wnt/planar cell polarity (PCP) pathway in this process in mammals.
In vertebrates, the PCP pathway has been shown to function during the convergent extension movements of gastrulation and neurulation, and in hair cell polarisation (reviewed by [5, 6]). The 'non-canonical Wnts', including Wnt5a, Wnt7a and Wnt11 are implicated in these processes [7–10]. Although the evidence linking Wnts with the PCP pathway is controversial, PCP signalling operates via Frizzleds (Fzs), and the membrane protein VanGogh-like 2 (Vangl2), while the cytoplasmic protein Scribble1 (Scrb1; which has been linked to apico-basal polarity) may also play a role. These components activate Dishevelled, which activates RhoA/Rho kinase (ROCK), and/or Rac and c-Jun amino-terminal kinase (JNK), leading to phosphorylation of molecules involved in cytoskeletal dynamics [11, 12]. The Wnt/PCP molecule Vangl2 can bind to Dishevelled, and can signal via both ROCK and JNK [13–16]. In addition, Wnt5a can activate JNK to influence convergent extension , and is required for the localisation of Vangl2 in cochlear hair cells . Thus, it is plausible that in FBM migration, JNK and ROCK form part of a signalling cascade containing Wnt5a, Fz, Vangl2, and other PCP components. Other downstream kinases that might function in FBM migration include myosin light chain kinase (MLCK), which is phosphorylated by ROCK and is implicated in cell migration (reviewed by ).
While recent (mainly loss-of-function) studies suggest that the Wnt/PCP pathway functions in FBM migration in the zebrafish [19–22], it is unclear which mechanisms operate in mammals (reviewed by ). In mice, there are few studies on the mechanisms of FBM migration, although vascular endothelial growth factor (VEGF) has been proposed as a chemoattractant . In this paper, we demonstrate a role of Wnts, other Wnt/PCP components and their downstream signalling pathways in the migration of FBM mammalian neurons.
Materials and methods
For appropriate gestational ages, we counted 0.5 days post-coitus as the formation of a vaginal plug on the following morning after matings. CD1 females were used as the wild-type strain. Vangl2, Scribble, Fz3 and Wnt5a homozygous mutants were obtained and genotyped as previously described [10, 15, 21, 25, 26]. Fz3 mutants were kindly provided by Dr Jeremy Nathans.
Hindbrain explant cultures
Entire hindbrain explants were cultured on millipore filters (Costar, Fisher, Loughborough, Leicestershire, UK) as previously described  and were supplemented with glial cell line-derived neurotrophic factor (R&D systems, Abingdon, Oxfordshire, UK). For chemoattraction affinity bead experiments, Cibacron blue 3AG agarose beads (Sigma-Aldrich, St. Louis, MO, USA) were soaked in phosphate-buffered saline (PBS; control) or in 100 μg/ml of either Wnt5a, Wnt7a, Semaphorin 3A (Sema3A; R&D systems) or VEGF (Peprotech Inc., London, UK) at 4°C overnight, before implantation into hindbrain explants. For the inhibitor experiments, explants were treated with culture medium containing either 10 μM JNK inhibitor (SP 600125, Tocris Bioscience, Bristol UK), 10 μM ROCK inhibitor (Y27632, Calbiochem, UK) or 10 μM MLCK inhibitor (ML-7, Calbiochem). In some cases, inhibitors were applied to hindbrain explants containing Wnt beads to test whether chemoattraction by beads was blocked. For inhibition of Wnt function, a cocktail of secreted frizzed-related proteins (SFRPs; 1, 2 and 3; 500 ng/ml; R&D systems) was applied to explants in the culture medium. Although binding specificities of SFRPs are not completely characterised, SFRP 1 and 2 can bind Wnt5a (for example, see ). A combination of SFRPs was applied to block a spectrum of putative Wnt-mediated interactions. After 2 days, explants were fixed with 4% paraformaldehyde for 1 hour and processed for immunohistochemistry.
Immunohistochemistry and scoring of FBM migration in explants
After fixation, explants were washed several times with PBS containing 1% Triton (PBT) then blocked in PBT containing 10% sheep serum for 30 minutes. Subsequently, the explants were incubated overnight at 4°C with anti-Islet1/2 antibody (4D5; 1:200; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), then washed with PBT for several hours, and afterwards incubated for 1 hour with Cy3 anti-mouse secondary antibody (1:800; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). Finally, the explants were washed with PBT and mounted on slides using Fluor-Save (Calbiochem).
Each explant was scored blind using one of the two following scoring systems. For bead implantation chemoattraction assays, explants were scored as 'attraction' if FBM neurons were deflected from their normal trajectory or as 'no attraction' if the FBM migration stream was unperturbed. Explants containing beads plus inhibitors were scored in the same way, focussing on whether FBM cells were deflected from the migration path, rather than on the migration as a whole. For explants treated with inhibitors, explants were scored on a scale of 1–3, ranging from loss of migration (1) to intermediate loss of migration (2) to normal migration (3). As some neurons had already reached r5 in most explants at the time of culture, we scored loss of migration (1) as a failure of neurons to reach r6 and/or a reduction in the number of neurons that had reached r5. Intermediate loss of migration was scored if explants manifest a phenotype between normal and loss of migration, that is, some neurons having reached r6 but less than in the normal case. In some experiments using inhibitors, the dorsal migration of trigeminal motor neurons in r2/3 was also quantified on a 1–3 scale. For each treatment, the percentage of explants in each category was calculated and compared statistically between control and treated groups (χ-squared test).
Quantification of FBM migration in explants and in Wnt5a mutant embryos
For experiments with control (PBS-treated) beads, Wnt5a-treated beads alone or with JNK or ROCK inhibitors, migration was quantified using the Scion image (NIH image) programme. Confocal images were rendered in black and white and inverted so that black pixels represented migrating neurons. For explants containing beads, a box was drawn containing the beads themselves (located at the r3/4 boundary) and encompassing r4 ipsilateral to the beads up to the midpoint of the floor plate (Additional file 1A). The number of pixels within the box was counted and the mean number of pixels under each condition was presented graphically. For explants without beads and cultured as controls or with inhibitors, a box was drawn over r4 bilaterally and over r6 bilaterally, containing the FBM neuron migratory stream (Additional file 1E). The ratio of r6 pixels/r4 pixels gives an indication of how successfully migration has occurred. The mean ratio was calculated under each condition. For Wnt5a mutant embryos and wild-type littermates, the width of the FBM migratory stream was measured at a dorsoventral midpoint using the Scion image programme to quantify the defect. Statistical comparisons were done using a t-test.
In situ hybridisation and immunohistochemistry
In situ hybridisation on whole-mounts or on cryosections of mouse embryos was performed as previously described [28, 29]. Tissues were hybridised with specific digoxigenin-labeled probes for Islet-1 (gift from Professor A Simeone, SEMM, Naples, Italy), Wnt 5a, Wnt 7a, EphA4 (gift from Dr U Drescher, MRC Centre of Developmental Neurobiology, London, UK), or Slit1 (gift from Dr M Tessier-Lavigne, Genentech, San Francisco, USA).
Wnt 5a and Wnt7a attract FBM neurons in a migration assay
In order to investigate the molecular mechanisms of FBM migration, we used a migration assay  in which E11.5 mouse hindbrains were isolated and cultured, flattened on filters, for 48 hours. Hindbrains were dissected out early on E11.5, as it was found that isolation of hindbrains on E10.5 led to poor tissue and motor neuron viability. Immunostaining of explants with anti-Islet-1/2 antibody at time 0 showed that most FBM neurons were located in r4, whereas a minority had migrated into r5 (Figure 1G). After 24 hours in vitro, many FBM neurons had reached r5 and some had reached r6 and started to turn laterally (Figure 1H). After 48 hours, FBM neurons had reached r6 and coalesced into a characteristic compact nucleus (Figure 1I), reflecting a similar or slightly later developmental stage than that observed using Islet-1 in situ hybridisation on E12.5 hindbrains (Figure 1F). Other branchiomotor neurons, such as those of the trigeminal nucleus, which undergo a lateral migration, were also visualised using Islet-1/2 immunostaining (data not shown).
Previous studies have shown that FBM neuron migration is, in part, dependent on VEGF binding to its neuropilin-1 (Npn-1) receptor, whereas the alternative Npn-1 ligand Sema3A plays no role . We therefore used VEGF and Sema3A-coated beads in this assay as positive and a negative controls, respectively. Our results confirmed that while VEGF acted as a chemoattractant, Sema3A had no effect (Figure 2D). These data show that Wnt5a and Wnt7a can chemoattract FBM neurons in hindbrain explants. In our assay, the magnitude of the effect is similar to that of VEGF.
Wnt5a and Wnt7a expression patterns in the hindbrain are consistent with a possible role as FBM guidance cues
FBM migration is severely disrupted in Vangl2 and Scribble mutants
By E13.5 in wild-type embryos (Figure 4B), FBM neurons had clustered in r6 while in both Vangl2 and Scribble mutants there was a highly abnormal distribution of FBM neurons, with the majority having migrated laterally to form an ectopic facial motor nucleus in dorsal r4 (Figure 4E, H). This migration mode is more characteristic of other populations of hindbrain branchiomotor neurons, for example, trigeminal . Another subpopulation of FBM neurons was located in the centre of the floor plate in r4 (Figure 4E, H). Some heterozygotes of both lines appeared normal, but in a few cases at E13.5 there were FBM neurons in rostral r5 that had diverged from the migratory stream, and the facial motor nucleus appeared less compact compared with wild-type embryos, occupying part of r5 as well as r6 (Figure 4I–K). These observations suggest that the normal caudal migratory path of FBM neurons had been transformed into a lateral or medial migration route in the absence of either of these two PCP genes (Figure 4G–I). The aberrant lateral FBM migration is reminiscent of that seen in Hoxb1 mutants at early developmental stages (E11.5), in which mis-specification of the FBM population results in a 'default state' of lateral migration, characteristic of branchiomotor neurons at other axial levels [2, 38]. The aberrant medial migration route suggests that some FBM neurons might fail to express receptors for Netrin-1 or Slit, which are normally involved in repelling cranial motor neurons from the floor plate [36, 39]. In Vangl2 and Scribble homozygous embryos, the lateral migration of other branchiomotor neuron populations was normal, for example, trigeminal (Figure 4E, H), suggesting that these genes specifically direct the caudal FBM migration. mRNA in situ hybridisation for Wnt5a in the Vangl2 mutant background showed a normal distribution of transcript, excluding the possibility that the mutant phenotype could be caused by the absence or a change in the expression of Wnt5a (Additional file 2C, D).
Attenuation of Wnt signalling in vitro and in vivo disrupts FBM migration
We also analysed Wnt7a and Wnt5a mutant mouse embryos, by performing mRNA in situ hybridisation for Islet-1 on E11.5 hindbrains. In Wnt7a mutant embryos, the distribution of FBM neurons closely resembled that in wild-type litter-mates, suggesting that there was no defect (data not shown). However, Wnt5a mutants showed a partial defect in the migration, with some divergent streams of migrating cells, resulting in a broader and less compact FBM nucleus extending into r5 as well as r6, suggesting that some of the FBM neurons had stalled (Figure 5D, E, G–I). This phenotype resembled the phenotype of heterozygous Vangl2 and Scribble mutant embryos (Figure 4J, K). Wnt5a heterozygous embryos showed no defects and were indistinguishable from wild-type embryos (Figure 5D; data not shown).
In order to quantify the Wnt5a mutant defect, we measured the width of the FBM migratory stream at a mid-point between the ventral edge of the migratory stream and the dorsal edge of the nucleus. This method revealed a significant increase in the width of the migratory stream in Wnt5a mutants compared with heterozygous embryos (Figure 5F), consistent with the decompaction of the nucleus observed. The phenotype that we observe in Wnt5a mutants suggests that Wnt5a plays a role in FBM migration in vivo, although we hypothesise that other Wnts might also play a role.
A candidate receptor to mediate the effect of Wnt in our system is Fz3, which is expressed in the mouse hindbrain at relevant developmental stages . Fz3 is involved in hair cell polarity in the mouse and its homologue, Fz3a, is implicated in FBM migration in the zebrafish [21, 42, 43]. We investigated the migration pattern of FBM neurons in Fz3 mutants  by Islet-1 in situ hybridisation as above. We found that there were striking defects in FBM migration in E11.5 and E14.5 homozygous Fz3 mutants compared with their wild-type or heterozygous littermates. At E11.5 the majority of FBM neurons had failed to migrate caudally out of r4 and remained close to the midline (Figure 5L, compare with Figure 5J). A reduced number of FBM neurons could be observed migrating caudally, but many neurons also formed streams migrating laterally in r4 and r5. At E14.5 the FBM nucleus was well-formed in wild-type embryos and was located in r6 (Figure 5K). In Fz3 mutants, an FBM nucleus was present in r6, but it was strongly reduced in size (Figure 5M). The presence of ectopic nuclei in dorsal r4 and r5 suggests that these originated from the dorsally migrating FBM neurons observed at E11.5. Thus, in Fz3 mutants, FBM migration shows severe defects consistent with the involvement of Fz signalling in this system. Attenuation of this pathway leads to many FBM neurons following a lateral 'default' migration pathway, in the manner of other hindbrain branchiomotor neuron subpopulations.
JNK and ROCK kinases signal during FBM migration and are required for the attractant effects of Wnts
To discover whether these inhibitors produced a general block of neuronal migration in the hindbrain, or had specific effects on the FBM caudal migration, we also assessed their effects on laterally migrating trigeminal (branchiomotor) neurons. JNK and ROCK inhibitors had modest effects in inhibiting lateral migration, though these effects were considerably less than those on FBM neurons' caudal migration (Additional file 3A, B). However, inhibition of MLCK produced effects that were more pronounced (45% of explants at grade 1) and equivalent to those on the FBM caudal migration (Additional file 3C). These data suggest that while MLCK plays a general role in cranial motor neuron migration, JNK and ROCK show specificity in functioning within the FBM caudal migration. As MLCK is a direct target of ROCK, this suggests that MLCK function in the dorsal migration might also be regulated by other upstream components. We also tested whether JNK and ROCK inhibitors blocked FBM neuron attraction by VEGF. Scoring of explants indicated that these inhibitors failed to block attraction by VEGF beads, indicating that JNK and ROCK are specific to Wnt-mediated FBM migration (Additional file 1C, D).
In view of the fact that Wnt5a is the most promising candidate to be an FBM chemoattractant in vivo, we next tested whether JNK or ROCK functioned downstream of Wnt5a, by applying inhibitors to explants containing implanted Wnt5a beads. The attractant effect of Wnt5a beads was specifically blocked by adding 10 μM of JNK inhibitor or of ROCK inhibitor (Figure 6J, K). This effect was further quantified by pixel counting (Materials and methods) of the number of FBM neurons in r4 adjacent to Wnt5a beads alone or in the presence of inhibitors. This showed a significant reduction in the number of FBM neurons adjacent to Wnt beads when explants were treated with inhibitors compared with those containing Wnt5a beads alone (Additional file 1B). This suggests that the effect of Wnt5a in FBM migration is mediated by the JNK and ROCK kinases.
We here propose that Wnt5a/7a act as guidance cues/chemoattractants in mammalian neuronal migration. Wnts have previously been implicated in neuronal migrations in Caenorhabditis elegans, where they act as chemorepellents [45, 46]. Although PCP components play a role in FBM migration in zebrafish [19–22], no Wnt ligands have so far been linked with the migration (reviewed by ). In the mouse, correlative data have showed that in Tbx20 mutant mice with aberrant FBM neuron migration, several Wnt/PCP components are down-regulated . Our study provides functional data suggesting a role for Wnt signalling via a non-canonical pathway, probably involving PCP components, in mammalian FBM migration.
Wnt5a/7a act as guidance cues/chemoattractants
In vertebrates, a role for Wnt5a in cell migration is indicated by the finding that down-regulation of Wnt5a impedes cell migration in a wound-healing assay . Our findings add to a growing body of evidence suggesting that the 'non-canonical Wnts', including Wnt5a, Wnt7a and Wnt11, function in aspects of PCP, including convergent extension movements (for example, [7, 8]), and cochlear hair cell orientation [9, 10]. Intriguingly, Wnt11 is also expressed in FBM neurons, and is absent in Tbx20 mutants . It is possible, therefore, that several Wnts collaborate during the migration. We found that Wnt7a mutant mice had no deficit in FBM migration, whereas in Wnt5a mutants, the migration pattern and nuclear formation were impaired. The relatively weak phenotype in Wnt5a mutants, however, suggests that whereas Wnt5a plays an important role, Wnt7a and/or other Wnts might play an adjunct role. This idea has a parallel in studies demonstrating that Wnt7a and Wnt5a both pattern cochlear hair cell polarisation, but only Wnt5a mutants show cochlear defects [9, 10]. In our system the more uniform expression pattern of Wnt7a might not rule out a possible role in the migration, as in zebrafish, several other Wnt/PCP components that play a functional role in FBM migration are widely expressed in the hindbrain. Interestingly, in the zebrafish, no Wnt ligands have so far been linked to the FBM migration (reviewed by ). It remains a formal possibility in the mouse hindbrain that, as has been suggested in other systems, activation of the PCP pathway might depend on a gradient of Fz activity without involving a Wnt ligand [5, 6].
Previous studies on axon guidance in vertebrates have pointed to a role for Wnts in chemoattraction and chemorepulsion of commissural axons and corticospinal axons, respectively, along the rostrocaudal axis of the spinal cord [26, 49]. In both cases Wnts (4 and 1/5a) were proposed to be distributed in a rostralhigh to caudallow gradient, the opposite polarity to that of Wnt5a expression in the hindbrain. It is likely, therefore, that local differences in Wnt gradients along the rostrocaudal axis, coupled with utilisation of different receptors/receptor complexes, dictate different cellular responses. In our proposed model, it is possible that endogenous SFRPs also play a role; SFRP2 is expressed in r4 in the mouse E10.5 hindbrain , and might enhance the caudal to rostral Wnt gradient by sequestering Wnt rostrally.
Another reason for the relatively modest phenotype in Wnt5a mutants is likely to be the role played by VEGF in FBM migration. VEGF is expressed in the floor plate and in a lateral stripe in the mouse hindbrain at E12.5, as well as in a group of cells lateral to the FBM nucleus . VEGF acts as a chemoattractant when presented on beads in an explant FBM migration assay  and the present study). VEGF and Wnt signalling are therefore likely to co-operate during the process of FBM migration. As Wnt5a is expressed in a rostro-caudal gradient and VEGF is expressed laterally, it may be that Wnts and VEGF play a role in the caudal and the lateral cell displacements, respectively. It is as yet unclear whether they utilise common signalling pathways; our experiments suggest that VEGF does not act via the JNK and ROCK kinases
The PCP pathway is implicated downstream of Wnts
It appears that at least one Fz protein, Fz3, functions in FBM migration. In Fz3 mutants, as in Vangl2 and Scribble mutants, the majority of FBM neurons migrated laterally (Figure 7A). Fz3 is globally expressed in the mouse hindbrain , and in zebrafish, Fz3a has been shown to act non-cell autonomously to control FBM migration . Although we cannot distinguish between a cell autonomous or non-cell autonomous mechanism of action of Fz3 in our system, the two mechanisms may not be mutually exclusive. Fz3 might function in chemoattraction of FBM neurons in a cell-autonomous manner, but a Fz activity gradient in the neuroepithelium might also play a role. This would not exclude the possibility that other Fzs might be involved. Fz4 and Fz7 can interact with Wnt5a, and Fz7 is expressed by FBM neurons [47, 55]. Other studies link the alternative Wnt receptor Ror2 to Wnt5a-induced cell migration . The possibility of an association between Fz and Vangl2 is also suggested by the observation that a multi-PDZ containing protein, MAGI-3, can bind Fz4 and Fz7 and Vangl2, providing the basis of a receptor complex for JNK signalling .
JNK and ROCK kinases play a role in FBM migration
Our investigation of downstream signalling in FBM migration by the use of inhibitors revealed a most striking role for JNK, both in the migration per se, and in the deflection of FBM neurons by the presentation of ectopic Wnt5a protein on beads. ROCK inhibition had a less striking effect on the migration pattern, but could also block the Wnt5a effect. Inhibition of MLCK disrupted FBM migration equivalently to ROCK inhibition, but also inhibited the dorsal migration of other hindbrain branchiomotor neurons, suggesting that it does not function specifically in the rostrocaudal migration route. Both JNK and ROCK are well-known to function downstream in the PCP pathway (reviewed by [12, 31]; see Figure 7B for schema). Wnt5a and the PCP pathway have also been linked to both JNK and ROCK in convergent extension movements in fish and frogs [17, 58–60]. However, in mammals, PCP downstream signalling pathways have been less clear. Whereas Wnt5a activates JNK in cultured mammalian cells [17, 55], a study focussing on convergent extension in the mouse found a role for RhoA-ROCK, but not JNK, downstream of Vangl2 .
We cannot completely rule out the possibility that JNK and ROCK function independently of the PCP pathway in our system. However, on balance, our findings suggest that both JNK and ROCK are important effectors of Wnt5a signalling in FBM neurons. By contrast, MLCK may play a role as a common target for several signalling pathways involved in the migration of branchiomotor (including FBM) neurons. A major target of MLCK is myosin II, which localises to the rear of the nucleus during neuronal migration [61, 62]; mice mutant for myosin IIB show defective facial motor nucleus formation [63, 64], consistent with our MLCK inhibitor studies. The downstream targets of JNK and ROCK phosphorylation in FBM neurons are unknown. Promising candidates are microtubule-associated proteins (MAPs); for example, known targets of JNK are the MAPs MAP1B and doublecortin [65, 66].
Finally, it will be intriguing for future studies to discover whether Wnt5a or other Wnts orchestrate neuronal migrations or axon projection patterns along the rostrocaudal axis of the hindbrain.
In this paper we demonstrate that the Wnt/PCP pathway functions in mammalian FBM migration via an attractant mechanism. Both Wnts are expressed in the hindbrain, and Wnt5a is expressed in a rostrallow to caudalhigh gradient consistent with a chemoattractant role. An in vivo role for Wnts is suggested by the finding that attenuation of endogenous Wnt signalling using SFRPs, or by genetic loss of function of Wnt5a, disrupts the FBM migration. Genetic deletion of the Wnt/PCP genes Fz3, Vangl2 and Scribble leads to severe disruption of FBM migration. We further show that the JNK and ROCK kinases are downstream components of Wnt signalling in FBM neurons. Our study is the first implicating Wnt/PCP signalling in mammalian FBM neuron migration.
c-Jun amino-terminal kinase
myosin light chain kinase
phosphate-buffered saline containing Triton-X100
planar cell polarity
secreted frizzed-related protein
Van Gogh-like 2
vascular endothelial growth factor.
We are grateful to Dr Dawn Savery and Dr Andrew Copp for initial help with obtaining Vangl2 and Scribble mutant mice. We thank Dr Lorenza Ciani and Dr Patricia Salinas for providing reagents, Wnt7a mutant mice and helpful advice. Thanks also to Dr Britta Eickholt for help with inhibitor studies, and to Dr Uwe Drescher for valuable discussions on the manuscript. We thank the Wellcome Trust very much for funding the work described in this article.
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