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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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Motor neurons migrate to ectopic positions in Npn-2  mice. (a,b) Dual immunostaining of transverse cryosections (20 μm) of E11.5 wild-type (a) or Npn-2  (b) littermate mouse embryo spinal cord with antibodies against HB9 (red) or neurofilament (NF; green) reveal numerous HB9-positive ectopic motor neurons located in the marginal zone and ventral nerve root in mutant but not wild-type embryo sections ((b) white arrows). Bar = 150 μm. (c) A quantitative analysis of the distribution of HB9-positive ectopic motor neurons along the rostro-caudal axis of Npn-2  (triangles) or heterozygous (squares) and wild-type (diamonds) littermate embryos indicates that the number of ectopic motor neurons is normal at forelimb level (red box) but peaks at hind limb level (yellow box). (d) A comparison of pooled and averaged number of HB9-positive ectopic motor neurons in the anterior (forelimb containing half) and the posterior (hindlimb containing half) of the trunk, for E12.5 Npn-2 wild-type, heterozygous and  littermate embryos (n = 4). Consistent with the quantitative analysis shown in (c), there is a significant increase in ectopic motor neurons in the hindlimb region of Npn-2  mice. **P ≤ 0.01; two-tailed t-test.
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Figure 3: Motor neurons migrate to ectopic positions in Npn-2 mice. (a,b) Dual immunostaining of transverse cryosections (20 μm) of E11.5 wild-type (a) or Npn-2 (b) littermate mouse embryo spinal cord with antibodies against HB9 (red) or neurofilament (NF; green) reveal numerous HB9-positive ectopic motor neurons located in the marginal zone and ventral nerve root in mutant but not wild-type embryo sections ((b) white arrows). Bar = 150 μm. (c) A quantitative analysis of the distribution of HB9-positive ectopic motor neurons along the rostro-caudal axis of Npn-2 (triangles) or heterozygous (squares) and wild-type (diamonds) littermate embryos indicates that the number of ectopic motor neurons is normal at forelimb level (red box) but peaks at hind limb level (yellow box). (d) A comparison of pooled and averaged number of HB9-positive ectopic motor neurons in the anterior (forelimb containing half) and the posterior (hindlimb containing half) of the trunk, for E12.5 Npn-2 wild-type, heterozygous and littermate embryos (n = 4). Consistent with the quantitative analysis shown in (c), there is a significant increase in ectopic motor neurons in the hindlimb region of Npn-2 mice. **P ≤ 0.01; two-tailed t-test.

Mentions: In order to test whether this mechanism was conserved in a mammalian species, we examined Npn-2 mice for evidence of ectopically positioned motor neurons. Npn-2 mutant mice have been analysed extensively in a number of studies of axon guidance phenotypes [13,25-27] and neuron migration [16,22,28] but defects in spinal motor neuron migration have not been reported. Yet we identified large numbers of ectopically positioned HB9-positive motor neuron cell bodies in Npn-2 mouse embryos (Figure 3b). Interestingly, this phenotype appeared to be confined to more caudal regions of the trunk, prompting an analysis of the incidence of ectopic motor neurons throughout the rostro-caudal axis. A typical result of such an analysis in serial cryosections taken throughout the trunk revealed ectopically positioned HB9-positive motor neurons were largely restricted to the hindlimb level (Figure 3c). At forelimb and thoracic levels there was no significant increase above the background level in wild-type littermate embryos. The pooled results of several E12.5 embryos analysed in this manner (n = 4 for each genotype) confirmed the presence of large numbers of ectopic motor neurons in the hindlimb region of Npn-2 mice (Figure 3d).


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)

Motor neurons migrate to ectopic positions in Npn-2  mice. (a,b) Dual immunostaining of transverse cryosections (20 μm) of E11.5 wild-type (a) or Npn-2  (b) littermate mouse embryo spinal cord with antibodies against HB9 (red) or neurofilament (NF; green) reveal numerous HB9-positive ectopic motor neurons located in the marginal zone and ventral nerve root in mutant but not wild-type embryo sections ((b) white arrows). Bar = 150 μm. (c) A quantitative analysis of the distribution of HB9-positive ectopic motor neurons along the rostro-caudal axis of Npn-2  (triangles) or heterozygous (squares) and wild-type (diamonds) littermate embryos indicates that the number of ectopic motor neurons is normal at forelimb level (red box) but peaks at hind limb level (yellow box). (d) A comparison of pooled and averaged number of HB9-positive ectopic motor neurons in the anterior (forelimb containing half) and the posterior (hindlimb containing half) of the trunk, for E12.5 Npn-2 wild-type, heterozygous and  littermate embryos (n = 4). Consistent with the quantitative analysis shown in (c), there is a significant increase in ectopic motor neurons in the hindlimb region of Npn-2  mice. **P ≤ 0.01; two-tailed t-test.
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Figure 3: Motor neurons migrate to ectopic positions in Npn-2 mice. (a,b) Dual immunostaining of transverse cryosections (20 μm) of E11.5 wild-type (a) or Npn-2 (b) littermate mouse embryo spinal cord with antibodies against HB9 (red) or neurofilament (NF; green) reveal numerous HB9-positive ectopic motor neurons located in the marginal zone and ventral nerve root in mutant but not wild-type embryo sections ((b) white arrows). Bar = 150 μm. (c) A quantitative analysis of the distribution of HB9-positive ectopic motor neurons along the rostro-caudal axis of Npn-2 (triangles) or heterozygous (squares) and wild-type (diamonds) littermate embryos indicates that the number of ectopic motor neurons is normal at forelimb level (red box) but peaks at hind limb level (yellow box). (d) A comparison of pooled and averaged number of HB9-positive ectopic motor neurons in the anterior (forelimb containing half) and the posterior (hindlimb containing half) of the trunk, for E12.5 Npn-2 wild-type, heterozygous and littermate embryos (n = 4). Consistent with the quantitative analysis shown in (c), there is a significant increase in ectopic motor neurons in the hindlimb region of Npn-2 mice. **P ≤ 0.01; two-tailed t-test.
Mentions: In order to test whether this mechanism was conserved in a mammalian species, we examined Npn-2 mice for evidence of ectopically positioned motor neurons. Npn-2 mutant mice have been analysed extensively in a number of studies of axon guidance phenotypes [13,25-27] and neuron migration [16,22,28] but defects in spinal motor neuron migration have not been reported. Yet we identified large numbers of ectopically positioned HB9-positive motor neuron cell bodies in Npn-2 mouse embryos (Figure 3b). Interestingly, this phenotype appeared to be confined to more caudal regions of the trunk, prompting an analysis of the incidence of ectopic motor neurons throughout the rostro-caudal axis. A typical result of such an analysis in serial cryosections taken throughout the trunk revealed ectopically positioned HB9-positive motor neurons were largely restricted to the hindlimb level (Figure 3c). At forelimb and thoracic levels there was no significant increase above the background level in wild-type littermate embryos. The pooled results of several E12.5 embryos analysed in this manner (n = 4 for each genotype) confirmed the presence of large numbers of ectopic motor neurons in the hindlimb region of Npn-2 mice (Figure 3d).

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