Planar cell polarity genes in motor axon guidance in the limb
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The assembly of neuronal circuits depends on the correct wiring of axons and dendrites. Studies in our laboratory revealed a critical role of seven-pass atypical cadherin Celsr3, a member of planar cell polarity (PCP) proteins, in the development of axonal tracts in the central nervous system, such as the anterior commissure, internal capsule and corticospinal tract. Celsr3 deficiency does not alter axonal growth, but affects axon guidance in cell-autonomous or non-cell-autonomous manners, causing axon stalling at intermediate targets or rerouting. Notably, all axon guidance defects in Celsr3−/− were observed in mice bearing mutations in the PCP gene Fzd3, and some errors were reported in mice with mutations of Vangl2, another PCP gene. Despite their unequivocally role, underlying molecular mechanisms remain elusive. Furthermore, their functions in the peripheral nervous system are still largely unexplored. Here we show that Celsr3 cooperates with Fzd3 in spinal motor neurons to mediate pathfinding of motor axons innervating the dorsal limb. Celsr3 is expressed in postmitotic neurons in the developing spinal cord. Specific inactivation of Celsr3 in spinal motor neurons severely perturbs peroneal nerve development, leading to absent innervation of the tibialis anterior muscle and stiff hindlimb. Deletion of Celsr3 affects neither the specification of motor neurons nor neuronal survival or neurite outgrowth. Celsr3-deficient axons of the peroneal nerve segregate from those of the tibial nerve but fail to extend dorsally, and they stall just after the branch point of the sciatic nerve. Mutant axons respond to repulsive ephrinA-EphA forward signaling and attractive glial cell–derived neurotrophic factor (GDNF). However, they are insensitive to attractive EphA-ephrinA reverse signaling. In transfected cells, Celsr3 immunoprecipitates with ephrinA2, ephrinA5, Ret, GDNF family receptor a1 (GFRa1) and Fzd3. The function of Celsr3 in motor axons is Fzd3 dependent but Vangl2 independent. Our results thus revealed the crucial roles of Celsr3 and Fzd3 in motor axon guidance, and provide evidence for the first time that the Celsr3-Fzd3 pathway interacts with EphA-ephrinA reverse signaling to guide motor axons in the hindlimb, which may help us better understand their molecular mechanisms of action.Keywords:
Commissure
Peripheral Nervous System
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Laminin is known to provide a highly permissive substratum and in some cases directional information for axon outgrowth in vitro. However, there is still little known about laminin function in guiding axons in vivo. We investigated the axon guidance role of laminin-alpha1 in the developing zebrafish nervous system. Analysis of zebrafish bashful (bal)/laminin-a1 mutants revealed multiple functions for laminin-alpha1 in the outgrowth and guidance of central nervous system (CNS) axons. Most CNS axon pathways are defective in bal embryos. Some axon types, including retinal ganglion cell axons, early forebrain axons, and hindbrain reticulospinal axons, make specific pathfinding errors, suggesting laminin-alpha1 is required for directional decisions. Other axon tracts are defasciculated or not fully extended in bal embryos, suggesting a function for laminin-alpha1 in regulating adhesion or providing a permissive substratum for growth. In addition, some neurons have excessively branched axons in bal, indicating a potential role for laminin-alpha1 in branching. In contrast to CNS axons, most peripheral axons appear normal in bal mutants. Our results, thus, reveal important and diverse functions for laminin-alpha1 in guiding developing axons in vivo.
Hindbrain
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Ephrin
Motor Control
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Hindlimb
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Type II spiral ganglion neurons provide afferent innervation to outer hair cells of the cochlea and are proposed to have nociceptive functions important for auditory function and homeostasis. These neurons are anatomically distinct from other classes of spiral ganglion neurons because they extend a peripheral axon beyond the inner hair cells that subsequently makes a distinct 90 degree turn toward the cochlear base. As a result, patterns of outer hair cell innervation are coordinated with the tonotopic organization of the cochlea. Previously, it was shown that peripheral axon turning is directed by a nonautonomous function of the core planar cell polarity (PCP) protein VANGL2. We demonstrate using mice of either sex that Fzd3 and Fzd6 similarly regulate axon turning, are functionally redundant with each other, and that Fzd3 genetically interacts with Vangl2 to guide this process. FZD3 and FZD6 proteins are asymmetrically distributed along the basolateral wall of cochlear-supporting cells, and are required to promote or maintain the asymmetric distribution of VANGL2 and CELSR1. These data indicate that intact PCP complexes formed between cochlear-supporting cells are required for the nonautonomous regulation of axon pathfinding. Consistent with this, in the absence of PCP signaling, peripheral axons turn randomly and often project toward the cochlear apex. Additional analyses of Porcn mutants in which WNT secretion is reduced suggest that noncanonical WNT signaling establishes or maintains PCP signaling in this context. A deeper understanding of these mechanisms is necessary for repairing auditory circuits following acoustic trauma or promoting cochlear reinnervation during regeneration-based deafness therapies. SIGNIFICANCE STATEMENT Planar cell polarity (PCP) signaling has emerged as a complementary mechanism to classical axon guidance in regulating axon track formation, axon outgrowth, and neuronal polarization. The core PCP proteins are also required for auditory circuit assembly, and coordinate hair cell innervation with the tonotopic organization of the cochlea. This is a non–cell-autonomous mechanism that requires the formation of PCP protein complexes between cochlear-supporting cells located along the trajectory of growth cone navigation. These findings are significant because they demonstrate how the fidelity of auditory circuit formation is ensured during development, and provide a mechanism by which PCP proteins may regulate axon outgrowth and guidance in the CNS.
Spiral ganglion
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The human brain contains more than 10 billion neurons that form over 10 trillion connections. The establishment of these connections during development requires axons to extend through the extracellular environment to their synaptic targets. This process of axon guidance is mediated by molecular cues in the extracellular matrix known as axon guidance molecules. Longitudinal axon tracts such as the striatonigral (SN), striatopallidal (SP), dopaminergic (mdDA) and serotonergic (5HT) pathways require molecular signals for their proper development. Impairment in their connectivity is implicated in Huntington’s diseases, Parkinson’s disease, drug addiction, depression and schizophrenia. Surprisingly little is known about the molecular events that underlie the establishment of these pathways. Wnt proteins are secreted molecules generally expressed in gradients along the anterior-posterior (A-P) axis of the developing nervous system inducing axon growth and guidance by binding axonal receptors such as Frizzleds and Ryk. This thesis explores a novel role for Wnts and their receptors in the A-P guidance of SP, SN, mdDA and 5HT axon tracts and for the first time establishes a role for Wnt/PCP signalling in the formation of these axon tracts. The SP and the SN axon tracts originate from the striatum and project to the substantia nigra retiulate (SNr) indirectly or directly, respectively. Our results show that SP and the SN axons grow alongside to the GP and that during this early phase of axon guidance the Frizzled3 (Fzd3) receptor is required for GP entry. Our findings are the first to demonstrate that during specific parts of their trajectories SP and SN axons use common molecular cues for navigating target structures such as the GP. mdDA axons grow in the rostral direction to form axon projections in the forebrain. We discovered that Wnt/PCP signalling dictates the rostral orientation of mdDA axons at the level of the midbrain. The core PCP receptors Fzd3, Vangl2 and Celsr3 were expressed in mdDA neurons and axons. Additionally, Wnt5a and Wnt7b were expressed in opposite gradients in the midbrain. Using collagen assays, we demonstrated that Wnt5a is a chemorepellent and Wnt7b a chemoattractant cue for mdDA axons. Serotonergic (5HT) neurons send their ascending axons to the forebrain and descending axons project to the spinal. 5HT neurons are born in the ventricular zone and migrate to the raphe by somal translocation. We found that for 5HT neurons, axon guidance appears to precede the final orientation of the soma. In wild-type animals, cell bodies of migrating, descending 5HT neurons initially point laterally but begin to reorient along the A–P axis as they approach their final lateral position, thereby obeying the direction of their axon projections. Our open book assay shows that the 5HT axons are responsive to Wnt4, Wnt5a and Wnt7a. Furthermore, they require core PCP receptors such as Fzd3, Celsr3 and Vangl2 to sense the Wnts. Overall our findings not only help to understand how these longitudinal axon connections are established but also provide a framework for understanding how these pathways may be changed in disease and could be altered in therapeutic settings.
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Myotome
Growth cone
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Ephrin
Ectodomain
Cleavage (geology)
Netrin
Growth cone
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Repulsive Eph forward signaling from limb-derived ephrins guides the axons of lateral motor column (LMC) motor neurons. LMC axons also express ephrinAs, while their EphA receptors are expressed in the limb mesenchyme. In vitro studies have suggested that reverse signaling from limb-derived EphA4 to axonal ephrinAs might result in attraction of LMC axons. However, genetic evidence for this function is lacking. Here we use the Dunn chamber turning assay to show that EphA proteins are chemoattractants and elicit fast turning responses in LMC neurons in vitro . Moreover, ectopic expression of EphA4 in chick hindlimb changes the limb trajectory of LMC axons. Nervous system-specific deletion of EphA4 in mice resulted in fewer LMC axon projection errors than the ubiquitous deletion of EphA4. Additionally, a signaling-incompetent EphA4 mutant partially rescued guidance errors in the hindlimb, suggesting that limb-derived EphA4 contributes to the establishment of LMC projections. In summary, we provide evidence for a role of EphA:ephrinA attractive reverse signaling in motor axon guidance and in vivo evidence of in-parallel forward Eph and reverse ephrin signaling function in the same neuronal population.
Ephrin
Limb bud
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During neural development, spinal motor axons extend in a precise manner from the ventral portion of the developing spinal cord to innervate muscle targets in the limb. Although classical studies in avians have characterized the cellular interactions that influence motor axon pathfinding to the limb, less is known about the molecular mechanisms that mediate this developmental event. Here, we examine the spatiotemporal distributions of the EphA4 receptor tyrosine kinase (RTK) and its cognate ligands, ephrin-A2 and ephrin-A5, on motor neurons, their axons and their pathways to the avian hindlimb to determine whether these molecules may influence axonal projections. The expression patterns of EphA4, ephrin-A2 and ephrin-A5 mRNAs and proteins are highly complex and appear to exhibit some overlap during motor axon outgrowth and pathfinding to the hindlimb, reminiscent of the co-expression of Eph RTKs and ephrins in the retinotectal system. EphA4, similar to the carbohydrate moiety polysialic acid, strikingly marks the main dorsal, but not ventral, nerve trunk after axon sorting at the limb plexus region. Our results suggest that EphA4 RTK and its ligands may influence axon fasciculation and the sorting of axons at the limb plexus, contributing to the correct dorsoventral organization of nerve branches in the hindlimb.
Ephrin
Limb bud
Growth cone
Hindlimb
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Rac1 is a critical regulator of cytoskeletal dynamics in multiple cell types. In the nervous system, it has been implicated in the control of cell proliferation, neuronal migration, and axon development. To systematically investigate the role of Rac1 in axon growth and guidance in the developing nervous system, we have examined the phenotypes associated with deleting Rac1 in the embryonic mouse forebrain, in cranial and spinal motor neurons, in cranial sensory and dorsal root ganglion neurons, and in the retina. We observe a widespread requirement for Rac1 in axon growth and guidance and a cell-autonomous defect in axon growth in Rac1 −/− motor neurons in culture. Neuronal death, presumably a secondary consequence of the axon growth and/or guidance defects, was observed in multiple locations. Following deletion of Rac1 in the forebrain, thalamocortical axons were misrouted inferiorly, with the majority projecting to the contralateral thalamus and a minority projecting ipsilaterally to the ventral cortex, a pattern of misrouting that is indistinguishable from the pattern previously observed in Frizzled3 −/− and Celsr3 −/− forebrains. In the limbs, motor-neuron-specific deletion of Rac1 produced a distinctive stalling of axons within the dorsal nerve of the hindlimb but a much milder loss of axons in the ventral hindlimb and forelimb nerves, a pattern that is virtually identical to the one previously observed in Frizzled3 −/− limbs. The similarities in axon growth and guidance phenotypes caused by Rac1, Frizzled3, and Celsr3 loss-of-function mutations suggest a mechanistic connection between tissue polarity/planar cell polarity signaling and Rac1-dependent cytoskeletal regulation.
Forebrain
Growth cone
Dorsal root ganglion
Peripheral Nervous System
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Citations (50)