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Boundary cap cells constrain spinal motor neuron somal migration at motor exit points by a semaphorin-plexin mechanism.

Bron R, Vermeren M, Kokot N, Andrews W, Little GE, Mitchell KJ, Cohen J - Neural Dev (2007)

Bottom Line: We conclude that semaphorin-mediated repellent interactions between boundary cap cells and immature spinal motor neurons regulates somal positioning by countering the drag exerted on motor neuron cell bodies by their axons as they emerge from the CNS at motor exit points.Our data support a model in which BC cell semaphorins signal through Npn-2 and/or Plexin-A2 receptors on motor neurons via a cytoplasmic effector, MICAL3, to trigger cytoskeletal reorganisation.This leads to the disengagement of somal migration from axon extension and the confinement of motor neuron cell bodies to the spinal cord.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Developmental Neurobiology, King's College London, Guy's Campus, London Bridge, London, SE1 1UL, UK. rbron@unimelb.edu.au

ABSTRACT

Background: In developing neurons, somal migration and initiation of axon outgrowth often occur simultaneously and are regulated in part by similar classes of molecules. When neurons reach their final destinations, however, somal translocation and axon extension are uncoupled. Insights into the mechanisms underlying this process of disengagement came from our study of the behaviour of embryonic spinal motor neurons following ablation of boundary cap cells. These are neural crest derivatives that transiently reside at motor exit points, central nervous system (CNS):peripheral nervous system (PNS) interfaces where motor axons leave the CNS. In the absence of boundary cap cells, motor neuron cell bodies migrate along their axons into the periphery, suggesting that repellent signals from boundary cap cells regulate the selective gating of somal migration and axon outgrowth at the motor exit point. Here we used RNA interference in the chick embryo together with analysis of mutant mice to identify possible boundary cap cell ligands, their receptors on motor neurons and cytoplasmic signalling molecules that control this process.

Results: We demonstrate that targeted knock down in motor neurons of Neuropilin-2 (Npn-2), a high affinity receptor for class 3 semaphorins, causes their somata to migrate to ectopic positions in ventral nerve roots. This finding was corroborated in Npn-2 mice, in which we identified motor neuron cell bodies in ectopic positions in the PNS. Our RNA interference studies further revealed a role for Plexin-A2, but not Plexin-A1 or Plexin-A4. We show that chick and mouse boundary cap cells express Sema3B and 3G, secreted semaphorins, and Sema6A, a transmembrane semaphorin. However, no increased numbers of ectopic motor neurons are found in Sema3B mouse embryos. In contrast, Sema6A mice display an ectopic motor neuron phenotype. Finally, knockdown of MICAL3, a downstream semaphorin/Plexin-A signalling molecule, in chick motor neurons led to their ectopic positioning in the PNS.

Conclusion: We conclude that semaphorin-mediated repellent interactions between boundary cap cells and immature spinal motor neurons regulates somal positioning by countering the drag exerted on motor neuron cell bodies by their axons as they emerge from the CNS at motor exit points. Our data support a model in which BC cell semaphorins signal through Npn-2 and/or Plexin-A2 receptors on motor neurons via a cytoplasmic effector, MICAL3, to trigger cytoskeletal reorganisation. This leads to the disengagement of somal migration from axon extension and the confinement of motor neuron cell bodies to the spinal cord.

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Expression of semaphorin receptors Npn-1 and Npn-2, and Plexin-A1, -A2, and -A4 by motor neurons in the chick embryo spinal cord. In situ hybridisations on transverse cryosections (20 μm) of chick embryo spinal cord (hindlimb level) from (b-f) HH stage 20 and (h-l) HH stage 23, show that Npn-1 (b,h), Npn-2 (c,i) and Plexin-A1 (d,j), Plexin-A2 (e,k), and Plexin-A4 (f,l) are all expressed by motor neurons. (a,g) Comparable sections from the same stage embryos immunolabelled with the motor neuron specific antibodies MNR2/islet2 (red) and counterstained with neurofilament antibody (NF; green). Bars = 50 μm.
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Figure 1: Expression of semaphorin receptors Npn-1 and Npn-2, and Plexin-A1, -A2, and -A4 by motor neurons in the chick embryo spinal cord. In situ hybridisations on transverse cryosections (20 μm) of chick embryo spinal cord (hindlimb level) from (b-f) HH stage 20 and (h-l) HH stage 23, show that Npn-1 (b,h), Npn-2 (c,i) and Plexin-A1 (d,j), Plexin-A2 (e,k), and Plexin-A4 (f,l) are all expressed by motor neurons. (a,g) Comparable sections from the same stage embryos immunolabelled with the motor neuron specific antibodies MNR2/islet2 (red) and counterstained with neurofilament antibody (NF; green). Bars = 50 μm.

Mentions: Previously, we proposed that neural crest BC cells at the motor exit point (MEP) express repellent signals that help confine motor neuron somata within the chick embryo spinal cord as their axons first emerge into the periphery [18]. This requires that candidate receptors for putative repellents should be expressed by motor neurons from embryonic day (E) 2 when the first motor axons exit the spinal cord at the MEP. Available expression data in mouse and chick embryos suggested that prominent amongst these were the multimeric surface receptors for secreted class 3 semaphorins, neuropilins and/or plexins [13-15]. We therefore analysed in detail expression of chick neuropilins and the three known members of the chick Plexin-A family, Plexin-A1, -A2 and -A4 (there is no chick Plexin-A3 orthologue [15]) during the appropriate period. Our results confirm and extend those of Mauti et al. [15], showing dynamic patterns of expression in spinal motor neurons, with Npn-1, and Plexin-A1 and -A2 already present in progenitors at Hamburger Hamilton (HH) stage 18 (not shown). We found that Npn-2 is also expressed in motor neurons (or their progenitors) as early as HH stage 18, and in rostral parts of the embryo and throughout the embryo by HH stage19 (not shown). At HH stage 20 all Npn and Plexin-A receptors are expressed by motor neurons to some extent (Figure 1b–f), with Npn-1 most prominently and distinctly expressed. Plexin-A1 and -A2 are widely expressed throughout the ventral spinal cord at this stage. Within the spinal cord, Npn-2 and Plexin-A4 expression is restricted to laterally positioned motor neurons.


Boundary cap cells constrain spinal motor neuron somal migration at motor exit points by a semaphorin-plexin mechanism.

Bron R, Vermeren M, Kokot N, Andrews W, Little GE, Mitchell KJ, Cohen J - Neural Dev (2007)

Expression of semaphorin receptors Npn-1 and Npn-2, and Plexin-A1, -A2, and -A4 by motor neurons in the chick embryo spinal cord. In situ hybridisations on transverse cryosections (20 μm) of chick embryo spinal cord (hindlimb level) from (b-f) HH stage 20 and (h-l) HH stage 23, show that Npn-1 (b,h), Npn-2 (c,i) and Plexin-A1 (d,j), Plexin-A2 (e,k), and Plexin-A4 (f,l) are all expressed by motor neurons. (a,g) Comparable sections from the same stage embryos immunolabelled with the motor neuron specific antibodies MNR2/islet2 (red) and counterstained with neurofilament antibody (NF; green). Bars = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2131750&req=5

Figure 1: Expression of semaphorin receptors Npn-1 and Npn-2, and Plexin-A1, -A2, and -A4 by motor neurons in the chick embryo spinal cord. In situ hybridisations on transverse cryosections (20 μm) of chick embryo spinal cord (hindlimb level) from (b-f) HH stage 20 and (h-l) HH stage 23, show that Npn-1 (b,h), Npn-2 (c,i) and Plexin-A1 (d,j), Plexin-A2 (e,k), and Plexin-A4 (f,l) are all expressed by motor neurons. (a,g) Comparable sections from the same stage embryos immunolabelled with the motor neuron specific antibodies MNR2/islet2 (red) and counterstained with neurofilament antibody (NF; green). Bars = 50 μm.
Mentions: Previously, we proposed that neural crest BC cells at the motor exit point (MEP) express repellent signals that help confine motor neuron somata within the chick embryo spinal cord as their axons first emerge into the periphery [18]. This requires that candidate receptors for putative repellents should be expressed by motor neurons from embryonic day (E) 2 when the first motor axons exit the spinal cord at the MEP. Available expression data in mouse and chick embryos suggested that prominent amongst these were the multimeric surface receptors for secreted class 3 semaphorins, neuropilins and/or plexins [13-15]. We therefore analysed in detail expression of chick neuropilins and the three known members of the chick Plexin-A family, Plexin-A1, -A2 and -A4 (there is no chick Plexin-A3 orthologue [15]) during the appropriate period. Our results confirm and extend those of Mauti et al. [15], showing dynamic patterns of expression in spinal motor neurons, with Npn-1, and Plexin-A1 and -A2 already present in progenitors at Hamburger Hamilton (HH) stage 18 (not shown). We found that Npn-2 is also expressed in motor neurons (or their progenitors) as early as HH stage 18, and in rostral parts of the embryo and throughout the embryo by HH stage19 (not shown). At HH stage 20 all Npn and Plexin-A receptors are expressed by motor neurons to some extent (Figure 1b–f), with Npn-1 most prominently and distinctly expressed. Plexin-A1 and -A2 are widely expressed throughout the ventral spinal cord at this stage. Within the spinal cord, Npn-2 and Plexin-A4 expression is restricted to laterally positioned motor neurons.

Bottom Line: We conclude that semaphorin-mediated repellent interactions between boundary cap cells and immature spinal motor neurons regulates somal positioning by countering the drag exerted on motor neuron cell bodies by their axons as they emerge from the CNS at motor exit points.Our data support a model in which BC cell semaphorins signal through Npn-2 and/or Plexin-A2 receptors on motor neurons via a cytoplasmic effector, MICAL3, to trigger cytoskeletal reorganisation.This leads to the disengagement of somal migration from axon extension and the confinement of motor neuron cell bodies to the spinal cord.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Developmental Neurobiology, King's College London, Guy's Campus, London Bridge, London, SE1 1UL, UK. rbron@unimelb.edu.au

ABSTRACT

Background: In developing neurons, somal migration and initiation of axon outgrowth often occur simultaneously and are regulated in part by similar classes of molecules. When neurons reach their final destinations, however, somal translocation and axon extension are uncoupled. Insights into the mechanisms underlying this process of disengagement came from our study of the behaviour of embryonic spinal motor neurons following ablation of boundary cap cells. These are neural crest derivatives that transiently reside at motor exit points, central nervous system (CNS):peripheral nervous system (PNS) interfaces where motor axons leave the CNS. In the absence of boundary cap cells, motor neuron cell bodies migrate along their axons into the periphery, suggesting that repellent signals from boundary cap cells regulate the selective gating of somal migration and axon outgrowth at the motor exit point. Here we used RNA interference in the chick embryo together with analysis of mutant mice to identify possible boundary cap cell ligands, their receptors on motor neurons and cytoplasmic signalling molecules that control this process.

Results: We demonstrate that targeted knock down in motor neurons of Neuropilin-2 (Npn-2), a high affinity receptor for class 3 semaphorins, causes their somata to migrate to ectopic positions in ventral nerve roots. This finding was corroborated in Npn-2 mice, in which we identified motor neuron cell bodies in ectopic positions in the PNS. Our RNA interference studies further revealed a role for Plexin-A2, but not Plexin-A1 or Plexin-A4. We show that chick and mouse boundary cap cells express Sema3B and 3G, secreted semaphorins, and Sema6A, a transmembrane semaphorin. However, no increased numbers of ectopic motor neurons are found in Sema3B mouse embryos. In contrast, Sema6A mice display an ectopic motor neuron phenotype. Finally, knockdown of MICAL3, a downstream semaphorin/Plexin-A signalling molecule, in chick motor neurons led to their ectopic positioning in the PNS.

Conclusion: We conclude that semaphorin-mediated repellent interactions between boundary cap cells and immature spinal motor neurons regulates somal positioning by countering the drag exerted on motor neuron cell bodies by their axons as they emerge from the CNS at motor exit points. Our data support a model in which BC cell semaphorins signal through Npn-2 and/or Plexin-A2 receptors on motor neurons via a cytoplasmic effector, MICAL3, to trigger cytoskeletal reorganisation. This leads to the disengagement of somal migration from axon extension and the confinement of motor neuron cell bodies to the spinal cord.

Show MeSH
Related in: MedlinePlus