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Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system.

Mauti O, Domanitskaya E, Andermatt I, Sadhu R, Stoeckli ET - Neural Dev (2007)

Bottom Line: Ablation of the boundary cap resulted in emigration of motoneurons from the ventral spinal cord along the ventral roots.Loss of either PlexinA4 or Sema6D function had an effect only at the dorsal root entry site but not at the ventral motor axon exit point.At the dorsal root entry site it organizes the segregation of dorsal roots.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Neuroscience, Institute of Zoology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Olivier.Mauti@zool.uzh.ch

ABSTRACT

Background: During spinal cord development, expression of chicken SEMAPHORIN6A (SEMA6A) is almost exclusively found in the boundary caps at the ventral motor axon exit point and at the dorsal root entry site. The boundary cap cells are derived from a population of late migrating neural crest cells. They form a transient structure at the transition zone between the peripheral nervous system (PNS) and the central nervous system (CNS). Ablation of the boundary cap resulted in emigration of motoneurons from the ventral spinal cord along the ventral roots. Based on its very restricted expression in boundary cap cells, we tested for a role of Sema6A as a gate keeper between the CNS and the PNS.

Results: Downregulation of Sema6A in boundary cap cells by in ovo RNA interference resulted in motoneurons streaming out of the spinal cord along the ventral roots, and in the failure of dorsal roots to form and segregate properly. PlexinAs interact with class 6 semaphorins and are expressed by both motoneurons and sensory neurons. Knockdown of PlexinA1 reproduced the phenotype seen after loss of Sema6A function both at the ventral motor exit point and at the dorsal root entry site of the lumbosacral spinal cord. Loss of either PlexinA4 or Sema6D function had an effect only at the dorsal root entry site but not at the ventral motor axon exit point.

Conclusion: Sema6A acts as a gate keeper between the PNS and the CNS both ventrally and dorsally. It is required for the clustering of boundary cap cells at the PNS/CNS interface and, thus, prevents motoneurons from streaming out of the ventral spinal cord. At the dorsal root entry site it organizes the segregation of dorsal roots.

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Downregulation of Sema6A in BCCs results in translocation of motoneurons out of the spinal cord. (a) In the absence of Sema6A from BCCs, motoneurons stream out of the ventral spinal cord and migrate along the ventral roots (arrows). The open arrow points to a motoneuron that is located in the ventral funiculus. (b) In control-treated embryos motoneurons along ventral roots or in the ventral funiculus were rarely seen. Motoneurons were identified by Isl-1 (red). An EGFP plasmid was co-injected with the dsRNA derived from SEMA6A. Axons were stained with an antibody against neurofilament (blue). Note that sensory neurons in the DRG (asterisk in (a, b)) are also stained by Isl-1. (c) Perturbation of Sema6B or Sema6D did not enhance the number of motoneurons in the periphery compared to control-treated embryos injected only with the plasmid encoding EGFP. Three asterisks indicate P < 0.0001 for the comparison between dsS6A and all other treatment groups. Values are given as mean ± standard error of mean. Bar: 50 μm.
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Figure 2: Downregulation of Sema6A in BCCs results in translocation of motoneurons out of the spinal cord. (a) In the absence of Sema6A from BCCs, motoneurons stream out of the ventral spinal cord and migrate along the ventral roots (arrows). The open arrow points to a motoneuron that is located in the ventral funiculus. (b) In control-treated embryos motoneurons along ventral roots or in the ventral funiculus were rarely seen. Motoneurons were identified by Isl-1 (red). An EGFP plasmid was co-injected with the dsRNA derived from SEMA6A. Axons were stained with an antibody against neurofilament (blue). Note that sensory neurons in the DRG (asterisk in (a, b)) are also stained by Isl-1. (c) Perturbation of Sema6B or Sema6D did not enhance the number of motoneurons in the periphery compared to control-treated embryos injected only with the plasmid encoding EGFP. Three asterisks indicate P < 0.0001 for the comparison between dsS6A and all other treatment groups. Values are given as mean ± standard error of mean. Bar: 50 μm.

Mentions: BCCs at the VMEP were shown to prevent the emigration of motoneurons from the ventral spinal cord [37]. The failure in BCC cluster formation after ablation of neural crest cells resulted in streams of motoneurons migrating along the axons of the ventral root. Because of the restricted expression of SEMA6A in BCCs, we set out to test whether Sema6A would be required for the role of BCCs as gate keepers between the CNS and the PNS. To this end, we used in ovo RNA interference (RNAi), our previously established method to induce loss-of-function phenotypes [48]. In ovo RNAi at HH12-14 efficiently targeted neural crest cells and resulted in downregulation of Sema6A but did not interfere with the expression of other family members of class 6 semaphorins (data not shown). Downregulation of Sema6A did indeed reproduce the phenotype seen after ablation of the BCCs (Figure 2) [37]. Groups of motoneurons identified by Isl-1 staining were found along the ventral roots in all HH25 embryos lacking Sema6A function (Figure 2a). Motoneurons exiting the spinal cord were seen, on average, in 40% of the sections from the lumbosacral spinal cord (range 25–54%). Single motoneurons leaving the ventral spinal cord were occasionally detected in control embryos (Figure 2b). However, cells did not emigrate in clusters as seen after downregulation of Sema6A, and the number of sections that contained motoneurons along the ventral roots was much smaller in control-treated embryos compared to embryos lacking Sema6A function. Downregulation of the other class 6 semaphorins, Sema6B (12%) and Sema6D (10%), did not significantly enhance emigration of motoneurons compared to control embryos expressing enhanced green fluorescent protein (EGFP; 8%). SEMA6D but not SEMA6B was found to be expressed in BCCs (IA and ES, unpublished observation). However, as seen for KROX20, expression started only after clustering of BCCs. SEMA6D was not found in BCCs that were still migrating (data not shown).


Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system.

Mauti O, Domanitskaya E, Andermatt I, Sadhu R, Stoeckli ET - Neural Dev (2007)

Downregulation of Sema6A in BCCs results in translocation of motoneurons out of the spinal cord. (a) In the absence of Sema6A from BCCs, motoneurons stream out of the ventral spinal cord and migrate along the ventral roots (arrows). The open arrow points to a motoneuron that is located in the ventral funiculus. (b) In control-treated embryos motoneurons along ventral roots or in the ventral funiculus were rarely seen. Motoneurons were identified by Isl-1 (red). An EGFP plasmid was co-injected with the dsRNA derived from SEMA6A. Axons were stained with an antibody against neurofilament (blue). Note that sensory neurons in the DRG (asterisk in (a, b)) are also stained by Isl-1. (c) Perturbation of Sema6B or Sema6D did not enhance the number of motoneurons in the periphery compared to control-treated embryos injected only with the plasmid encoding EGFP. Three asterisks indicate P < 0.0001 for the comparison between dsS6A and all other treatment groups. Values are given as mean ± standard error of mean. Bar: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Downregulation of Sema6A in BCCs results in translocation of motoneurons out of the spinal cord. (a) In the absence of Sema6A from BCCs, motoneurons stream out of the ventral spinal cord and migrate along the ventral roots (arrows). The open arrow points to a motoneuron that is located in the ventral funiculus. (b) In control-treated embryos motoneurons along ventral roots or in the ventral funiculus were rarely seen. Motoneurons were identified by Isl-1 (red). An EGFP plasmid was co-injected with the dsRNA derived from SEMA6A. Axons were stained with an antibody against neurofilament (blue). Note that sensory neurons in the DRG (asterisk in (a, b)) are also stained by Isl-1. (c) Perturbation of Sema6B or Sema6D did not enhance the number of motoneurons in the periphery compared to control-treated embryos injected only with the plasmid encoding EGFP. Three asterisks indicate P < 0.0001 for the comparison between dsS6A and all other treatment groups. Values are given as mean ± standard error of mean. Bar: 50 μm.
Mentions: BCCs at the VMEP were shown to prevent the emigration of motoneurons from the ventral spinal cord [37]. The failure in BCC cluster formation after ablation of neural crest cells resulted in streams of motoneurons migrating along the axons of the ventral root. Because of the restricted expression of SEMA6A in BCCs, we set out to test whether Sema6A would be required for the role of BCCs as gate keepers between the CNS and the PNS. To this end, we used in ovo RNA interference (RNAi), our previously established method to induce loss-of-function phenotypes [48]. In ovo RNAi at HH12-14 efficiently targeted neural crest cells and resulted in downregulation of Sema6A but did not interfere with the expression of other family members of class 6 semaphorins (data not shown). Downregulation of Sema6A did indeed reproduce the phenotype seen after ablation of the BCCs (Figure 2) [37]. Groups of motoneurons identified by Isl-1 staining were found along the ventral roots in all HH25 embryos lacking Sema6A function (Figure 2a). Motoneurons exiting the spinal cord were seen, on average, in 40% of the sections from the lumbosacral spinal cord (range 25–54%). Single motoneurons leaving the ventral spinal cord were occasionally detected in control embryos (Figure 2b). However, cells did not emigrate in clusters as seen after downregulation of Sema6A, and the number of sections that contained motoneurons along the ventral roots was much smaller in control-treated embryos compared to embryos lacking Sema6A function. Downregulation of the other class 6 semaphorins, Sema6B (12%) and Sema6D (10%), did not significantly enhance emigration of motoneurons compared to control embryos expressing enhanced green fluorescent protein (EGFP; 8%). SEMA6D but not SEMA6B was found to be expressed in BCCs (IA and ES, unpublished observation). However, as seen for KROX20, expression started only after clustering of BCCs. SEMA6D was not found in BCCs that were still migrating (data not shown).

Bottom Line: Ablation of the boundary cap resulted in emigration of motoneurons from the ventral spinal cord along the ventral roots.Loss of either PlexinA4 or Sema6D function had an effect only at the dorsal root entry site but not at the ventral motor axon exit point.At the dorsal root entry site it organizes the segregation of dorsal roots.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Neuroscience, Institute of Zoology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Olivier.Mauti@zool.uzh.ch

ABSTRACT

Background: During spinal cord development, expression of chicken SEMAPHORIN6A (SEMA6A) is almost exclusively found in the boundary caps at the ventral motor axon exit point and at the dorsal root entry site. The boundary cap cells are derived from a population of late migrating neural crest cells. They form a transient structure at the transition zone between the peripheral nervous system (PNS) and the central nervous system (CNS). Ablation of the boundary cap resulted in emigration of motoneurons from the ventral spinal cord along the ventral roots. Based on its very restricted expression in boundary cap cells, we tested for a role of Sema6A as a gate keeper between the CNS and the PNS.

Results: Downregulation of Sema6A in boundary cap cells by in ovo RNA interference resulted in motoneurons streaming out of the spinal cord along the ventral roots, and in the failure of dorsal roots to form and segregate properly. PlexinAs interact with class 6 semaphorins and are expressed by both motoneurons and sensory neurons. Knockdown of PlexinA1 reproduced the phenotype seen after loss of Sema6A function both at the ventral motor exit point and at the dorsal root entry site of the lumbosacral spinal cord. Loss of either PlexinA4 or Sema6D function had an effect only at the dorsal root entry site but not at the ventral motor axon exit point.

Conclusion: Sema6A acts as a gate keeper between the PNS and the CNS both ventrally and dorsally. It is required for the clustering of boundary cap cells at the PNS/CNS interface and, thus, prevents motoneurons from streaming out of the ventral spinal cord. At the dorsal root entry site it organizes the segregation of dorsal roots.

Show MeSH
Related in: MedlinePlus