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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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shRNA mediated selective knockdown of Npn-2 or Plexin-A2 mRNAs in the chick ventral spinal cord induces ectopic positioning of motor neurons. (a-f) Confocal micrographs of transverse vibratome sections (75 μm) of HH stage 24 embryo spinal cord 2 days after electroporation in the ventral neural tube. MNR2/Islet2 positive ectopic motor neurons (red; white arrows) are found in embryos electroporated with shRNA-EGFP vectors specific for Npn-2 (a,b) and Plexin-A2 (e,f), but not for Plexin-A1 (c,d). The presence of the shRNA vector is indicated by EGFP expression (green) such that MNR2/Islet2 positive motor neuron somata in ectopic positions are yellow (arrows in (a,e)). Bar = 150 μm. (g) Histogram showing percentage of HH stage 24 embryo sections containing dual labelled EGFP and MNR2/Islet2 positive ectopic motor neurons after ventral electroporations at HH stage 12–15 with shRNA-EGFP vectors targeting Npn-1, Npn-2, Plexin-A1, Plexin-A2 and Plexin-A4, or EGFP control vector. Only those shRNA constructs targeting Npn-2 and Plexin-A2 induced ectopic positioning of motor neurons. ***P < 0.001; two-tailed t-test.
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Figure 2: shRNA mediated selective knockdown of Npn-2 or Plexin-A2 mRNAs in the chick ventral spinal cord induces ectopic positioning of motor neurons. (a-f) Confocal micrographs of transverse vibratome sections (75 μm) of HH stage 24 embryo spinal cord 2 days after electroporation in the ventral neural tube. MNR2/Islet2 positive ectopic motor neurons (red; white arrows) are found in embryos electroporated with shRNA-EGFP vectors specific for Npn-2 (a,b) and Plexin-A2 (e,f), but not for Plexin-A1 (c,d). The presence of the shRNA vector is indicated by EGFP expression (green) such that MNR2/Islet2 positive motor neuron somata in ectopic positions are yellow (arrows in (a,e)). Bar = 150 μm. (g) Histogram showing percentage of HH stage 24 embryo sections containing dual labelled EGFP and MNR2/Islet2 positive ectopic motor neurons after ventral electroporations at HH stage 12–15 with shRNA-EGFP vectors targeting Npn-1, Npn-2, Plexin-A1, Plexin-A2 and Plexin-A4, or EGFP control vector. Only those shRNA constructs targeting Npn-2 and Plexin-A2 induced ectopic positioning of motor neurons. ***P < 0.001; two-tailed t-test.

Mentions: We quantified the effect of shRNA-EGFP electroporation, compared to that of EGFP only, by counting ectopically positioned, EGFP-positive motor neurons. These were localised outside the spinal cord, along the ventral roots, and were characterised by their large soma linked to both trailing and leading EGFP-positive processes. Their identities were confirmed by dual labelling with antibodies to the transcription factors Islet-2 and motor neuron restricted protein (MNR)2. In our initial experiments with Npn-2 specific shRNA, we consistently observed ectopic EGFP/Islet-2/MNR-2 positive motor neurons in presumptive white matter and ventral roots (Figure 2a,b), but not with Npn-1 specific shRNA (Figure 2g). We subsequently generated four more vectors targeting Npn-2 and screened these for the potential to reduce Npn-2 expression in a co-expression assay [19]. One of these constructs, Npn-2 E shRNA, was as efficient as Npn-2 B shRNA in reducing Npn-2 expression (Additional file 2) and also consistently induced ectopic motor neurons, when electroporated in the ventral neural tube (Additional file 3).


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)

shRNA mediated selective knockdown of Npn-2 or Plexin-A2 mRNAs in the chick ventral spinal cord induces ectopic positioning of motor neurons. (a-f) Confocal micrographs of transverse vibratome sections (75 μm) of HH stage 24 embryo spinal cord 2 days after electroporation in the ventral neural tube. MNR2/Islet2 positive ectopic motor neurons (red; white arrows) are found in embryos electroporated with shRNA-EGFP vectors specific for Npn-2 (a,b) and Plexin-A2 (e,f), but not for Plexin-A1 (c,d). The presence of the shRNA vector is indicated by EGFP expression (green) such that MNR2/Islet2 positive motor neuron somata in ectopic positions are yellow (arrows in (a,e)). Bar = 150 μm. (g) Histogram showing percentage of HH stage 24 embryo sections containing dual labelled EGFP and MNR2/Islet2 positive ectopic motor neurons after ventral electroporations at HH stage 12–15 with shRNA-EGFP vectors targeting Npn-1, Npn-2, Plexin-A1, Plexin-A2 and Plexin-A4, or EGFP control vector. Only those shRNA constructs targeting Npn-2 and Plexin-A2 induced ectopic positioning of motor neurons. ***P < 0.001; two-tailed t-test.
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Figure 2: shRNA mediated selective knockdown of Npn-2 or Plexin-A2 mRNAs in the chick ventral spinal cord induces ectopic positioning of motor neurons. (a-f) Confocal micrographs of transverse vibratome sections (75 μm) of HH stage 24 embryo spinal cord 2 days after electroporation in the ventral neural tube. MNR2/Islet2 positive ectopic motor neurons (red; white arrows) are found in embryos electroporated with shRNA-EGFP vectors specific for Npn-2 (a,b) and Plexin-A2 (e,f), but not for Plexin-A1 (c,d). The presence of the shRNA vector is indicated by EGFP expression (green) such that MNR2/Islet2 positive motor neuron somata in ectopic positions are yellow (arrows in (a,e)). Bar = 150 μm. (g) Histogram showing percentage of HH stage 24 embryo sections containing dual labelled EGFP and MNR2/Islet2 positive ectopic motor neurons after ventral electroporations at HH stage 12–15 with shRNA-EGFP vectors targeting Npn-1, Npn-2, Plexin-A1, Plexin-A2 and Plexin-A4, or EGFP control vector. Only those shRNA constructs targeting Npn-2 and Plexin-A2 induced ectopic positioning of motor neurons. ***P < 0.001; two-tailed t-test.
Mentions: We quantified the effect of shRNA-EGFP electroporation, compared to that of EGFP only, by counting ectopically positioned, EGFP-positive motor neurons. These were localised outside the spinal cord, along the ventral roots, and were characterised by their large soma linked to both trailing and leading EGFP-positive processes. Their identities were confirmed by dual labelling with antibodies to the transcription factors Islet-2 and motor neuron restricted protein (MNR)2. In our initial experiments with Npn-2 specific shRNA, we consistently observed ectopic EGFP/Islet-2/MNR-2 positive motor neurons in presumptive white matter and ventral roots (Figure 2a,b), but not with Npn-1 specific shRNA (Figure 2g). We subsequently generated four more vectors targeting Npn-2 and screened these for the potential to reduce Npn-2 expression in a co-expression assay [19]. One of these constructs, Npn-2 E shRNA, was as efficient as Npn-2 B shRNA in reducing Npn-2 expression (Additional file 2) and also consistently induced ectopic motor neurons, when electroporated in the ventral neural tube (Additional file 3).

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