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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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Genetic ablation of Sema6A in BC cells leads to ectopic migration of motor neurons. (a) In situ hybridisation shows expression of Sema6A in BC cells at the MEP (black arrows) in transverse cryosections of E12 mouse embryos. Expression of egr2/krox-20 in an adjacent section (a') confirms the signal in (a) corresponds to BC cells. Bar = 100 μm. (b,c) Dual immunostaining of transverse cryosections of E13.5 Sema6A mouse embryos. Compared with wild-type littermates (b) where HB9 positive motor neuron somata (red) are exclusively confined to the ventral spinal cord, in Sema6A  embryos (c) many motor neurons can be seen in ectopic positions in the neurofilament-positive (green) presumptive white matter and ventral nerve roots. Bar = 100 μm. (d) A quantitative analysis of the distribution of HB9-positive ectopic motor neurons along the rostro-caudal axis of E13.5 mouse spinal cord shows a distinct peak at hindlimb level (yellow box) but not forelimb level (red box) in  (triangles) compared with heterozygous (squares) and wild-type (diamonds) embryo littermates. (e) A comparison of the cumulative counts of HB9 positive ectopic motor neurons in sections from posterior trunk (hindlimb containing) region of E13.5 Sema6A wild-type, heterozygous and  embryos. Consistent with the quantitative analysis shown in (d), there is a significant increase (p = 0.03; two-tail t-test) in ectopic motor neurons in the hindlimb region of Sema6A  mice (n = 4 each). (f,g) In situ hybridisation for egr2/krox-20 on transverse cryosections (30 μm) of E11.5 mouse embryos at hindlimb level. The results show no obvious difference in Egr2 expression at the DREZ or MEP in wild-type (f) or Sema6A  mice (g), indicating that formation of boundary caps is not perturbed by genetic ablation of Sema6A. Bar = 100 μm. *P ≤ 0.05; two-tailed t-test
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Figure 6: Genetic ablation of Sema6A in BC cells leads to ectopic migration of motor neurons. (a) In situ hybridisation shows expression of Sema6A in BC cells at the MEP (black arrows) in transverse cryosections of E12 mouse embryos. Expression of egr2/krox-20 in an adjacent section (a') confirms the signal in (a) corresponds to BC cells. Bar = 100 μm. (b,c) Dual immunostaining of transverse cryosections of E13.5 Sema6A mouse embryos. Compared with wild-type littermates (b) where HB9 positive motor neuron somata (red) are exclusively confined to the ventral spinal cord, in Sema6A embryos (c) many motor neurons can be seen in ectopic positions in the neurofilament-positive (green) presumptive white matter and ventral nerve roots. Bar = 100 μm. (d) A quantitative analysis of the distribution of HB9-positive ectopic motor neurons along the rostro-caudal axis of E13.5 mouse spinal cord shows a distinct peak at hindlimb level (yellow box) but not forelimb level (red box) in (triangles) compared with heterozygous (squares) and wild-type (diamonds) embryo littermates. (e) A comparison of the cumulative counts of HB9 positive ectopic motor neurons in sections from posterior trunk (hindlimb containing) region of E13.5 Sema6A wild-type, heterozygous and embryos. Consistent with the quantitative analysis shown in (d), there is a significant increase (p = 0.03; two-tail t-test) in ectopic motor neurons in the hindlimb region of Sema6A mice (n = 4 each). (f,g) In situ hybridisation for egr2/krox-20 on transverse cryosections (30 μm) of E11.5 mouse embryos at hindlimb level. The results show no obvious difference in Egr2 expression at the DREZ or MEP in wild-type (f) or Sema6A mice (g), indicating that formation of boundary caps is not perturbed by genetic ablation of Sema6A. Bar = 100 μm. *P ≤ 0.05; two-tailed t-test

Mentions: The finding of sema6A expression in chick motor exit point BC cells led us to examine expression in mouse. At E11.5 a distinct Sema6A signal is seen in motor exit point BC cells (Figure 6a, black arrowheads), in addition to prominent expression in the neural tube ventricular zone. Co-labelling with Egr2 (Krox-20) in an adjacent section (Figure 6a') confirmed expression was in BC cells. This prompted us to look for evidence of ectopic motor neuron positioning in Sema6A mice [33]. Like in Npn-2 mice, here we found numerous ectopic motor neurons in ventral nerve roots (Figure 6b,c). Also, in common with Npn-2 mice, HB9-positive ectopic motor neurons are most prevalent at the level of the hindlimb (Figure 6d,e). An effect on neural crest migration and on the formation of boundary caps could account for this phenotype. However, as judged by Egr2 expression, there was no difference between Sema6A mutants and wild-type littermates (Figure 6f,g) in the arrangement of BC cells at the MEP and DREZ.


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)

Genetic ablation of Sema6A in BC cells leads to ectopic migration of motor neurons. (a) In situ hybridisation shows expression of Sema6A in BC cells at the MEP (black arrows) in transverse cryosections of E12 mouse embryos. Expression of egr2/krox-20 in an adjacent section (a') confirms the signal in (a) corresponds to BC cells. Bar = 100 μm. (b,c) Dual immunostaining of transverse cryosections of E13.5 Sema6A mouse embryos. Compared with wild-type littermates (b) where HB9 positive motor neuron somata (red) are exclusively confined to the ventral spinal cord, in Sema6A  embryos (c) many motor neurons can be seen in ectopic positions in the neurofilament-positive (green) presumptive white matter and ventral nerve roots. Bar = 100 μm. (d) A quantitative analysis of the distribution of HB9-positive ectopic motor neurons along the rostro-caudal axis of E13.5 mouse spinal cord shows a distinct peak at hindlimb level (yellow box) but not forelimb level (red box) in  (triangles) compared with heterozygous (squares) and wild-type (diamonds) embryo littermates. (e) A comparison of the cumulative counts of HB9 positive ectopic motor neurons in sections from posterior trunk (hindlimb containing) region of E13.5 Sema6A wild-type, heterozygous and  embryos. Consistent with the quantitative analysis shown in (d), there is a significant increase (p = 0.03; two-tail t-test) in ectopic motor neurons in the hindlimb region of Sema6A  mice (n = 4 each). (f,g) In situ hybridisation for egr2/krox-20 on transverse cryosections (30 μm) of E11.5 mouse embryos at hindlimb level. The results show no obvious difference in Egr2 expression at the DREZ or MEP in wild-type (f) or Sema6A  mice (g), indicating that formation of boundary caps is not perturbed by genetic ablation of Sema6A. Bar = 100 μm. *P ≤ 0.05; two-tailed t-test
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Figure 6: Genetic ablation of Sema6A in BC cells leads to ectopic migration of motor neurons. (a) In situ hybridisation shows expression of Sema6A in BC cells at the MEP (black arrows) in transverse cryosections of E12 mouse embryos. Expression of egr2/krox-20 in an adjacent section (a') confirms the signal in (a) corresponds to BC cells. Bar = 100 μm. (b,c) Dual immunostaining of transverse cryosections of E13.5 Sema6A mouse embryos. Compared with wild-type littermates (b) where HB9 positive motor neuron somata (red) are exclusively confined to the ventral spinal cord, in Sema6A embryos (c) many motor neurons can be seen in ectopic positions in the neurofilament-positive (green) presumptive white matter and ventral nerve roots. Bar = 100 μm. (d) A quantitative analysis of the distribution of HB9-positive ectopic motor neurons along the rostro-caudal axis of E13.5 mouse spinal cord shows a distinct peak at hindlimb level (yellow box) but not forelimb level (red box) in (triangles) compared with heterozygous (squares) and wild-type (diamonds) embryo littermates. (e) A comparison of the cumulative counts of HB9 positive ectopic motor neurons in sections from posterior trunk (hindlimb containing) region of E13.5 Sema6A wild-type, heterozygous and embryos. Consistent with the quantitative analysis shown in (d), there is a significant increase (p = 0.03; two-tail t-test) in ectopic motor neurons in the hindlimb region of Sema6A mice (n = 4 each). (f,g) In situ hybridisation for egr2/krox-20 on transverse cryosections (30 μm) of E11.5 mouse embryos at hindlimb level. The results show no obvious difference in Egr2 expression at the DREZ or MEP in wild-type (f) or Sema6A mice (g), indicating that formation of boundary caps is not perturbed by genetic ablation of Sema6A. Bar = 100 μm. *P ≤ 0.05; two-tailed t-test
Mentions: The finding of sema6A expression in chick motor exit point BC cells led us to examine expression in mouse. At E11.5 a distinct Sema6A signal is seen in motor exit point BC cells (Figure 6a, black arrowheads), in addition to prominent expression in the neural tube ventricular zone. Co-labelling with Egr2 (Krox-20) in an adjacent section (Figure 6a') confirmed expression was in BC cells. This prompted us to look for evidence of ectopic motor neuron positioning in Sema6A mice [33]. Like in Npn-2 mice, here we found numerous ectopic motor neurons in ventral nerve roots (Figure 6b,c). Also, in common with Npn-2 mice, HB9-positive ectopic motor neurons are most prevalent at the level of the hindlimb (Figure 6d,e). An effect on neural crest migration and on the formation of boundary caps could account for this phenotype. However, as judged by Egr2 expression, there was no difference between Sema6A mutants and wild-type littermates (Figure 6f,g) in the arrangement of BC cells at the MEP and DREZ.

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