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1.  Regulation of neuronal migration by Dchs1-Fat4 planar cell polarity 
Current biology : CB  2014;24(14):1620-1627.
Planar-cell polarity (PCP) describes the polarisation of cell structures and behaviors within the plane of a tissue. PCP is essential for the generation of tissue architecture during embryogenesis and for post-natal growth and tissue repair, yet how it is oriented to coordinate cell polarity remains poorly understood [1]. In Drosophila, PCP is mediated via the Frizzled-Flamingo (Fz-PCP) and Dachsous-Fat (Fat-PCP) pathways [1-3]. Fz-PCP is conserved in vertebrates but an understanding in vertebrates of whether and how Fat-PCP polarizes cells, and its relationship to Fz-PCP signaling, is lacking. Mutations in human FAT4 and DCHS1 cause Van Maldergem syndrome, characterized by severe neuronal abnormalities indicative of altered neuronal migration [4]. Here, we investigate the role and mechanisms of Fat-PCP during neuronal migration using the murine facial branchiomotor neurons (FBM) as a model. We find that Fat4 and Dchs1, key components of Fat-PCP signaling, are expressed in complementary gradients and are required for the collective tangential migration of FBM and for their PCP. Fat4 and Dchs1 are required intrinsically within the FBM and extrinsically within the neuroepithelium. Remarkably, Fat-PCP and Fz-PCP regulate FBM migration along orthogonal axes. Disruption of the Dchs1 gradients by mosaic inactivation of Dchs1 alters FBM polarity and migration. This study implies that PCP in vertebrates can be regulated via gradients of Fat4 and Dchs1 expression, which establish intracellular polarity across FBM cells during their migration. Our results also identify Fat-PCP as a novel neuronal guidance system, and reveal that Fat-PCP and Fz-PCP can act along orthogonal axes.
PMCID: PMC4193925  PMID: 24998526
2.  Facial Motor Neuron Migration Advances 
Current opinion in neurobiology  2013;23(6):10.1016/j.conb.2013.09.001.
During development, the migration of specific neuronal subtypes is required for the correct establishment of neural circuits. In mice and zebrafish, facial branchiomotor (FBM) neurons undergo a tangential migration from rhombomere 4 caudally through the hindbrain. Recent advances in the field have capitalized on genetic studies in zebrafish and mouse, and high-resolution time-lapse imaging in zebrafish. Planar cell polarity signaling has emerged as a critical conserved factor in FBM neuron migration, functioning both within the neurons and their environment. In zebrafish, migration depends on specialized ‘pioneer’ neurons to lead follower FBM neurons through the hindbrain, and on interactions with structural components including pre-laid axon tracts and the basement membrane. Despite fundamental conservation, species-specific differences in migration mechanisms are being uncovered.
PMCID: PMC3852894  PMID: 24090878
3.  Central Topography of Cranial Motor Nuclei Controlled by Differential Cadherin Expression 
Current Biology  2014;24(21):2541-2547.
Neuronal nuclei are prominent, evolutionarily conserved features of vertebrate central nervous system (CNS) organization [1]. Nuclei are clusters of soma of functionally related neurons and are located in highly stereotyped positions. Establishment of this CNS topography is critical to neural circuit assembly. However, little is known of either the cellular or molecular mechanisms that drive nucleus formation during development, a process termed nucleogenesis [2–5]. Brainstem motor neurons, which contribute axons to distinct cranial nerves and whose functions are essential to vertebrate survival, are organized exclusively as nuclei. Cranial motor nuclei are composed of two main classes, termed branchiomotor/visceromotor and somatomotor [6]. Each of these classes innervates evolutionarily distinct structures, for example, the branchial arches and eyes, respectively. Additionally, each class is generated by distinct progenitor cell populations and is defined by differential transcription factor expression [7, 8]; for example, Hb9 distinguishes somatomotor from branchiomotor neurons. We characterized the time course of cranial motornucleogenesis, finding that despite differences in cellular origin, segregation of branchiomotor and somatomotor nuclei occurs actively, passing through a phase of each being intermingled. We also found that differential expression of cadherin cell adhesion family members uniquely defines each motor nucleus. We show that cadherin expression is critical to nucleogenesis as its perturbation degrades nucleus topography predictably.
•Cranial motor nucleogenesis occurs through an active process of segregation•Differential cadherin expression defines cranial motor nuclei•Cadherin expression drives specificity of cranial motor nucleus segregation•Cadherin expression does not affect cranial motor neuron migration
Astick et al. show that differential cadherin cell adhesion molecule expression defines brainstem motor neurons during their coalescence into discrete motor nuclei. No two motor nuclei express the same combination of cadherins. They demonstrate that the specificity of this expression is critical to the segregation of brainstem motor nuclei.
PMCID: PMC4228048  PMID: 25308074
4.  Tar DNA-binding protein-43 (TDP-43) regulates axon growth in vitro and in vivo☆ 
Neurobiology of Disease  2014;65(100):25-34.
Intracellular inclusions of the TAR-DNA binding protein 43 (TDP-43) have been reported in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD-TDP). Rare mutations in TARDBP have been linked to both ALS and FTD-TDP suggesting that TDP-43 dysfunction is mechanistic in causing disease. TDP-43 is a predominantly nuclear protein with roles in regulating RNA transcription, splicing, stability and transport. In ALS, TDP-43 aberrantly accumulates in the cytoplasm of motor neurons where it forms aggregates. However it has until recently been unclear whether the toxic effects of TDP-43 involve recruitment to motor axons, and what effects this might have on axonal growth and integrity. Here we use chick embryonic motor neurons, in vivo and in vitro, to model the acute effects of TDP-43. We show that wild-type and two TDP-43 mutant proteins cause toxicity in chick embryonic motor neurons in vivo. Moreover, TDP-43 is increasingly mislocalised to axons over time in vivo, axon growth to peripheral targets is truncated, and expression of neurofilament-associated antigen is reduced relative to control motor neurons. In primary spinal motor neurons in vitro, a progressive translocation of TDP-43 to the cytoplasm occurs over time, similar to that observed in vivo. This coincides with the appearance of cytoplasmic aggregates, a reduction in the axonal length, and cellular toxicity, which was most striking for neurons expressing TDP-43 mutant forms. These observations suggest that the capacity of spinal motor neurons to produce and maintain an axon is compromised by dysregulation of TDP-43 and that the disruption of cytoskeletal integrity may play a role in the pathogenesis of ALS and FTD-TDP.
•TDP-43 causes neurotoxicity in the chick embryo spinal cord.•Mutant TDP-43 forms nuclear inclusions and mis-localises to the cytoplasm.•TDP-43 leads to down-regulation of neurofilament-associated antigen.•TDP-43 causes premature truncation and de-fasciculation of axons in vivo.•TDP-43 causes cytoplasmic aggregation and reduced neurite outgrowth in vitro.
PMCID: PMC3988849  PMID: 24423647
TDP-43; ALS; FTD-TDP; Motor neuron; Chick embryo; Axon growth cytoskeleton; Cytoplasmic mislocalisation; Neurotoxicity
5.  α2-Chimaerin Regulates a Key Axon Guidance Transition during Development of the Oculomotor Projection 
The Journal of Neuroscience  2013;33(42):16540-16551.
The ocular motor system consists of three nerves which innervate six muscles to control eye movements. In humans, defective development of this system leads to eye movement disorders, such as Duane Retraction Syndrome, which can result from mutations in the α2-chimaerin signaling molecule. We have used the zebrafish to model the role of α2-chimaerin during development of the ocular motor system. We first mapped ocular motor spatiotemporal development, which occurs between 24 and 72 h postfertilization (hpf), with the oculomotor nerve following an invariant sequence of growth and branching to its muscle targets. We identified 52 hpf as a key axon guidance “transition,” when oculomotor axons reach the orbit and select their muscle targets. Live imaging and quantitation showed that, at 52 hpf, axons undergo a switch in behavior, with striking changes in the dynamics of filopodia. We tested the role of α2-chimaerin in this guidance process and found that axons expressing gain-of-function α2-chimaerin isoforms failed to undergo the 52 hpf transition in filopodial dynamics, leading to axon stalling. α2-chimaerin loss of function led to ecotopic and misguided branching and hypoplasia of oculomotor axons; embryos had defective eye movements as measured by the optokinetic reflex. Manipulation of chimaerin signaling in oculomotor neurons in vitro led to changes in microtubule stability. These findings demonstrate that a correct level of α2-chimaerin signaling is required for key oculomotor axon guidance decisions, and provide a zebrafish model for Duane Retraction Syndrome.
PMCID: PMC3797374  PMID: 24133258
6.  Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II 
Neural Development  2010;5:16.
In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A). It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear.
In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK) and myosin light chain kinase (MLCK), which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points.
Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.
PMCID: PMC2907369  PMID: 20569485
7.  Wnt activity guides facial branchiomotor neuron migration, and involves the PCP pathway and JNK and ROCK kinases 
Neural Development  2009;4:7.
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.
PMCID: PMC2654884  PMID: 19210786
8.  Human CHN1 mutations hyperactivate α2-chimaerin and cause Duane’s retraction syndrome 
Science (New York, N.Y.)  2008;321(5890):839-843.
The RacGAP molecule α2-chimaerin is implicated in neuronal signaling pathways required for precise guidance of developing corticospinal axons. We now demonstrate that a variant of Duane’s retraction syndrome, a congenital eye movement disorder in which affected individuals show aberrant development of axon projections to the extraocular muscles, can result from gain-of-function heterozygous missense mutations in CHN1 that increase α2-chimaerin RacGAP activity in vitro. A subset of mutations enhances α2-chimaerin membrane translocation and/or α2-chimaerin’s previously unrecognized ability to form a complex with itself. In ovo expression of mutant CHN1 alters the development of ocular motor axons. These data demonstrate that human CHN1 mutations can hyperactivate α2-chimaerin and result in aberrant cranial motor neuron development.
PMCID: PMC2593867  PMID: 18653847

Results 1-8 (8)