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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 knockdown of Sema6A in the neural crest, but not in the ventral spinal cord, induces ectopic motorneurons. (a,b) Confocal micrographs of vibratome sections of an HH stage 25 embryo, two days after electroporation in the neural crest with a Sema-6A shRNA-EGFP (green). Embryos were wholemount dual immunostained with antibodies against MNR2 and Islet2 (red) and counterstained with anti-neurofilament (NF, blue). MNR2 and Islet2-positive motor neurons (white arrowheads) can be seen in ectopic positions in the ventral roots but only when accompanied by a large number of Sema-6A shRNA expressing-EGFP-positive (green) neural crest derivatives. Note that EGFP-labelled cells are restricted to neural crest derivatives in the ventral root and that motor neurons, including those in ectopic positions are EGFP-negative. The small amount of EGFP labelling of structures in a ventro-medial position in the spinal cord corresponds to the axon projections of labelled commissural neurons in the dorsal spinal cord. Bar = 100 μm. (c) Quantitative analysis of the prevalence of ectopic motor neurons after electroporation with Sema-6A shRNA-EGFP targeted towards either the ventral neural tube (NT) or dorsal neural tube (neural crest, NC) shows a distinct effect only for neural crest-electroporated embryos. ***P < 0.001; two-tailed t-test. Conversely, knockdown of Npn-2 expression in the neural crest does not induce ectopic motor neurons, unlike knockdown of Npn-2 in the ventral neural tube, as shown earlier (Figure 2). (d) Confocal micrograph of a transverse cryosection (20 μm) immunolabelled with cad7(red) and neurofilament (blue) of an HH23 embryo 2 days after neural crest electroporation of Sema6A shRNA. GFP-positive neural crest cells targeted with Sema-6A shRNA populate the dorsal root ganglion (DRG), the DREZ and the MEP where they express high level of cad7 (red; white arrows), indicating that Sema6A shRNA does not interfere with boundary cap formation.
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Figure 7: shRNA mediated knockdown of Sema6A in the neural crest, but not in the ventral spinal cord, induces ectopic motorneurons. (a,b) Confocal micrographs of vibratome sections of an HH stage 25 embryo, two days after electroporation in the neural crest with a Sema-6A shRNA-EGFP (green). Embryos were wholemount dual immunostained with antibodies against MNR2 and Islet2 (red) and counterstained with anti-neurofilament (NF, blue). MNR2 and Islet2-positive motor neurons (white arrowheads) can be seen in ectopic positions in the ventral roots but only when accompanied by a large number of Sema-6A shRNA expressing-EGFP-positive (green) neural crest derivatives. Note that EGFP-labelled cells are restricted to neural crest derivatives in the ventral root and that motor neurons, including those in ectopic positions are EGFP-negative. The small amount of EGFP labelling of structures in a ventro-medial position in the spinal cord corresponds to the axon projections of labelled commissural neurons in the dorsal spinal cord. Bar = 100 μm. (c) Quantitative analysis of the prevalence of ectopic motor neurons after electroporation with Sema-6A shRNA-EGFP targeted towards either the ventral neural tube (NT) or dorsal neural tube (neural crest, NC) shows a distinct effect only for neural crest-electroporated embryos. ***P < 0.001; two-tailed t-test. Conversely, knockdown of Npn-2 expression in the neural crest does not induce ectopic motor neurons, unlike knockdown of Npn-2 in the ventral neural tube, as shown earlier (Figure 2). (d) Confocal micrograph of a transverse cryosection (20 μm) immunolabelled with cad7(red) and neurofilament (blue) of an HH23 embryo 2 days after neural crest electroporation of Sema6A shRNA. GFP-positive neural crest cells targeted with Sema-6A shRNA populate the dorsal root ganglion (DRG), the DREZ and the MEP where they express high level of cad7 (red; white arrows), indicating that Sema6A shRNA does not interfere with boundary cap formation.

Mentions: Since at later stages in the mouse Sema6A is expressed not only in BC cells but also in spinal motor neurons [14] (data not shown), it could not be excluded that the effect on motor neuron positioning in Sema6A mice was due to its absence from motor neurons. Therefore, we next tested in chick embryos the effects of targeted knock down of Sema6A, either ventrally in motor neurons, or dorsally in the neural crest (Additional file 1). We found a significant increase in ectopic motor neurons, but only when Sema6A shRNA was introduced in the neural crest (Figure 7a,b), not in the ventral neural tube (Figure 7c). Knockdown of Npn-2 in the neural crest or ventral neural tube had the converse effect, with ectopic motor neurons found only after expression of Npn-2 shRNA in the ventral neural tube (Figure 7c). Finally, we analysed the effect on boundary cap formation of Sema6A knockdown in the crest. The data show that Sema6A shRNA-expressing neural crest cells are normally positioned at the MEP and these correspond to cad7-positive BC cells (Figure 7d, white arrows). Together, these results support the idea that the effect of Sema6A loss of function on motor neuron positioning is explained by loss of its expression in BC cells, disrupting a putative interaction with 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)

shRNA mediated knockdown of Sema6A in the neural crest, but not in the ventral spinal cord, induces ectopic motorneurons. (a,b) Confocal micrographs of vibratome sections of an HH stage 25 embryo, two days after electroporation in the neural crest with a Sema-6A shRNA-EGFP (green). Embryos were wholemount dual immunostained with antibodies against MNR2 and Islet2 (red) and counterstained with anti-neurofilament (NF, blue). MNR2 and Islet2-positive motor neurons (white arrowheads) can be seen in ectopic positions in the ventral roots but only when accompanied by a large number of Sema-6A shRNA expressing-EGFP-positive (green) neural crest derivatives. Note that EGFP-labelled cells are restricted to neural crest derivatives in the ventral root and that motor neurons, including those in ectopic positions are EGFP-negative. The small amount of EGFP labelling of structures in a ventro-medial position in the spinal cord corresponds to the axon projections of labelled commissural neurons in the dorsal spinal cord. Bar = 100 μm. (c) Quantitative analysis of the prevalence of ectopic motor neurons after electroporation with Sema-6A shRNA-EGFP targeted towards either the ventral neural tube (NT) or dorsal neural tube (neural crest, NC) shows a distinct effect only for neural crest-electroporated embryos. ***P < 0.001; two-tailed t-test. Conversely, knockdown of Npn-2 expression in the neural crest does not induce ectopic motor neurons, unlike knockdown of Npn-2 in the ventral neural tube, as shown earlier (Figure 2). (d) Confocal micrograph of a transverse cryosection (20 μm) immunolabelled with cad7(red) and neurofilament (blue) of an HH23 embryo 2 days after neural crest electroporation of Sema6A shRNA. GFP-positive neural crest cells targeted with Sema-6A shRNA populate the dorsal root ganglion (DRG), the DREZ and the MEP where they express high level of cad7 (red; white arrows), indicating that Sema6A shRNA does not interfere with boundary cap formation.
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Figure 7: shRNA mediated knockdown of Sema6A in the neural crest, but not in the ventral spinal cord, induces ectopic motorneurons. (a,b) Confocal micrographs of vibratome sections of an HH stage 25 embryo, two days after electroporation in the neural crest with a Sema-6A shRNA-EGFP (green). Embryos were wholemount dual immunostained with antibodies against MNR2 and Islet2 (red) and counterstained with anti-neurofilament (NF, blue). MNR2 and Islet2-positive motor neurons (white arrowheads) can be seen in ectopic positions in the ventral roots but only when accompanied by a large number of Sema-6A shRNA expressing-EGFP-positive (green) neural crest derivatives. Note that EGFP-labelled cells are restricted to neural crest derivatives in the ventral root and that motor neurons, including those in ectopic positions are EGFP-negative. The small amount of EGFP labelling of structures in a ventro-medial position in the spinal cord corresponds to the axon projections of labelled commissural neurons in the dorsal spinal cord. Bar = 100 μm. (c) Quantitative analysis of the prevalence of ectopic motor neurons after electroporation with Sema-6A shRNA-EGFP targeted towards either the ventral neural tube (NT) or dorsal neural tube (neural crest, NC) shows a distinct effect only for neural crest-electroporated embryos. ***P < 0.001; two-tailed t-test. Conversely, knockdown of Npn-2 expression in the neural crest does not induce ectopic motor neurons, unlike knockdown of Npn-2 in the ventral neural tube, as shown earlier (Figure 2). (d) Confocal micrograph of a transverse cryosection (20 μm) immunolabelled with cad7(red) and neurofilament (blue) of an HH23 embryo 2 days after neural crest electroporation of Sema6A shRNA. GFP-positive neural crest cells targeted with Sema-6A shRNA populate the dorsal root ganglion (DRG), the DREZ and the MEP where they express high level of cad7 (red; white arrows), indicating that Sema6A shRNA does not interfere with boundary cap formation.
Mentions: Since at later stages in the mouse Sema6A is expressed not only in BC cells but also in spinal motor neurons [14] (data not shown), it could not be excluded that the effect on motor neuron positioning in Sema6A mice was due to its absence from motor neurons. Therefore, we next tested in chick embryos the effects of targeted knock down of Sema6A, either ventrally in motor neurons, or dorsally in the neural crest (Additional file 1). We found a significant increase in ectopic motor neurons, but only when Sema6A shRNA was introduced in the neural crest (Figure 7a,b), not in the ventral neural tube (Figure 7c). Knockdown of Npn-2 in the neural crest or ventral neural tube had the converse effect, with ectopic motor neurons found only after expression of Npn-2 shRNA in the ventral neural tube (Figure 7c). Finally, we analysed the effect on boundary cap formation of Sema6A knockdown in the crest. The data show that Sema6A shRNA-expressing neural crest cells are normally positioned at the MEP and these correspond to cad7-positive BC cells (Figure 7d, white arrows). Together, these results support the idea that the effect of Sema6A loss of function on motor neuron positioning is explained by loss of its expression in BC cells, disrupting a putative interaction with 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