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Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons.

Poliak S, Morales D, Croteau LP, Krawchuk D, Palmesino E, Morton S, Cloutier JF, Charron F, Dalva MB, Ackerman SL, Kao TJ, Kania A - Elife (2015)

Bottom Line: We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors.Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals.Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Columbia University, New York, United States.

ABSTRACT
During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

No MeSH data available.


Related in: MedlinePlus

Characterization of siRNA-mediated Unc5c knockdown and Unc5c overexpression in LMC neurons in chicken.(A) Detection of Isl1, Foxp1, GFP, and Unc5c in chick embryos electroporated with [Unc5c]siRNA and GFP. (B) Quantification of the reduction in expression levels of Unc5c in LMC neurons after in ovo electroporation of [Unc5c]siRNA. Note a significant decrease (p<0.001) in Unc5c expression levels by 50% upon [Unc5c]siRNA and GFP but not GFP-only electroporation. Number of embryos quantified: n = 3 for both groups. (C, D) The total number of LMC neurons and the proportion of medial and lateral LMC are normal after [Unc5]siRNA electroporation. Number of embryos quantified: n = 4 for all groups. (E) Equal proportion of medial and lateral LMC neurons electroporated with either GFP only or [Unc5c]siRNA and GFP. Number of embryos quantified: n = 4 for all groups. (F) The expression of EphA4 and EphB1 is not altered after [Unc5]siRNA electroporation. (G) Detection of Isl1, Foxp1, GFP, and mouse Unc5c in chick embryos expressing mUnc5c and GFP. (H, I) The total number of LMC neurons and the proportion of medial and lateral LMC are normal after mUnc5c and GFP electroporation. Number of embryos quantified: n = 4 for all groups. (J) Equal proportion of medial and lateral LMC neurons electroporated with either GFP only or mUnc5c and GFP. Number of embryos quantified: n = 4 for all groups. (K) The expression of EphA4 and EphB1 is not altered after mUnc5c and GFP electroporation. Number of embryos quantified: n = 4 for all groups. (L) Detection of GFP, mUnc5c, mEphb2, and Foxp1 in chick embryos expressing normal (top) or low (1/5th) (bottom) concentrations of Ephb2-GFP and Unc5c plasmids. (M) Quantification of the ratio of the ectopic expression levels of low and normal concentration of GFP, Unc5c, and EphB2 in chick embryos. Number of embryos quantified: n = 6 for all groups. (N) Analysis of lateral LMC motor axon projections in chick embryos electroporated with Unc5c, Ephb2, Unc5c and Ephb2, or GFP expression plasmids (all at low concentrations). A retrograde tracer (HRP, blue) was injected in the ventral forelimb of HH st. 29/30 chick embryos followed by detection of Lhx1 (red) to identify lateral LMC neurons. Insets show examples of magnified HRP+ backfilled cells that are Lhx1+ (in low Unc5c + low Ephb2 co-electroporation) or Lhx1- (in all other conditions). (O) Quantification of retrogradely labeled lateral LMC axon projections. The graph depicts the mean percentage ± SD of electroporated (GFP+) and HRP+ backfilled motor neurons that express the lateral LMC marker Lhx1 after a ventral limb injection. N ≥ 3 embryos. HRP, horseradish peroxidase; LMC, lateral motor column; error bars = SD; *** = p<0.001; n.s. = not significant; statistical significance computed using Mann-Whitney U test (B–E, H–J, M), or Fisher’s exact test on raw numbers (O); all values are mean ± SD. Scale bars: (A, G, L) 56 μm; (F, K) 145 μm; (N) 40 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.011
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fig4s1: Characterization of siRNA-mediated Unc5c knockdown and Unc5c overexpression in LMC neurons in chicken.(A) Detection of Isl1, Foxp1, GFP, and Unc5c in chick embryos electroporated with [Unc5c]siRNA and GFP. (B) Quantification of the reduction in expression levels of Unc5c in LMC neurons after in ovo electroporation of [Unc5c]siRNA. Note a significant decrease (p<0.001) in Unc5c expression levels by 50% upon [Unc5c]siRNA and GFP but not GFP-only electroporation. Number of embryos quantified: n = 3 for both groups. (C, D) The total number of LMC neurons and the proportion of medial and lateral LMC are normal after [Unc5]siRNA electroporation. Number of embryos quantified: n = 4 for all groups. (E) Equal proportion of medial and lateral LMC neurons electroporated with either GFP only or [Unc5c]siRNA and GFP. Number of embryos quantified: n = 4 for all groups. (F) The expression of EphA4 and EphB1 is not altered after [Unc5]siRNA electroporation. (G) Detection of Isl1, Foxp1, GFP, and mouse Unc5c in chick embryos expressing mUnc5c and GFP. (H, I) The total number of LMC neurons and the proportion of medial and lateral LMC are normal after mUnc5c and GFP electroporation. Number of embryos quantified: n = 4 for all groups. (J) Equal proportion of medial and lateral LMC neurons electroporated with either GFP only or mUnc5c and GFP. Number of embryos quantified: n = 4 for all groups. (K) The expression of EphA4 and EphB1 is not altered after mUnc5c and GFP electroporation. Number of embryos quantified: n = 4 for all groups. (L) Detection of GFP, mUnc5c, mEphb2, and Foxp1 in chick embryos expressing normal (top) or low (1/5th) (bottom) concentrations of Ephb2-GFP and Unc5c plasmids. (M) Quantification of the ratio of the ectopic expression levels of low and normal concentration of GFP, Unc5c, and EphB2 in chick embryos. Number of embryos quantified: n = 6 for all groups. (N) Analysis of lateral LMC motor axon projections in chick embryos electroporated with Unc5c, Ephb2, Unc5c and Ephb2, or GFP expression plasmids (all at low concentrations). A retrograde tracer (HRP, blue) was injected in the ventral forelimb of HH st. 29/30 chick embryos followed by detection of Lhx1 (red) to identify lateral LMC neurons. Insets show examples of magnified HRP+ backfilled cells that are Lhx1+ (in low Unc5c + low Ephb2 co-electroporation) or Lhx1- (in all other conditions). (O) Quantification of retrogradely labeled lateral LMC axon projections. The graph depicts the mean percentage ± SD of electroporated (GFP+) and HRP+ backfilled motor neurons that express the lateral LMC marker Lhx1 after a ventral limb injection. N ≥ 3 embryos. HRP, horseradish peroxidase; LMC, lateral motor column; error bars = SD; *** = p<0.001; n.s. = not significant; statistical significance computed using Mann-Whitney U test (B–E, H–J, M), or Fisher’s exact test on raw numbers (O); all values are mean ± SD. Scale bars: (A, G, L) 56 μm; (F, K) 145 μm; (N) 40 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.011

Mentions: Given the prominent function of ephrins in motor axon guidance, we next investigated the possibility that Netrin and ephrin signals co-operate in LMC neurons. To do this we focused on the function of Unc5c and EphB2, an ephrin-B receptor guiding medial LMC axons in vivo (Luria et al., 2008). We first tested whether chicken Unc5c is required in vivo by reducing its expression through [Unc5c]siRNA and GFP plasmid electroporation of presumptive LMC neurons at HH st. 17–19. Analysis of the proportion of electroporated LMC axons entering the dorsal versus ventral limb nerves at HH st. 26 showed that in embryos electroporated with [Unc5c]siRNA, 72% of GFP+ axons were present in the dorsal limb nerve, compared with 52% in GFP electroporated controls (Figure 4A,B; Figure 4—figure supplement 1; p<0.01), demonstrating that chicken Unc5c is required for the fidelity of LMC trajectory choice.10.7554/eLife.10841.010Figure 4.Co-operation between Unc5c and EphB2 receptors in LMC trajectory selection. 


Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons.

Poliak S, Morales D, Croteau LP, Krawchuk D, Palmesino E, Morton S, Cloutier JF, Charron F, Dalva MB, Ackerman SL, Kao TJ, Kania A - Elife (2015)

Characterization of siRNA-mediated Unc5c knockdown and Unc5c overexpression in LMC neurons in chicken.(A) Detection of Isl1, Foxp1, GFP, and Unc5c in chick embryos electroporated with [Unc5c]siRNA and GFP. (B) Quantification of the reduction in expression levels of Unc5c in LMC neurons after in ovo electroporation of [Unc5c]siRNA. Note a significant decrease (p<0.001) in Unc5c expression levels by 50% upon [Unc5c]siRNA and GFP but not GFP-only electroporation. Number of embryos quantified: n = 3 for both groups. (C, D) The total number of LMC neurons and the proportion of medial and lateral LMC are normal after [Unc5]siRNA electroporation. Number of embryos quantified: n = 4 for all groups. (E) Equal proportion of medial and lateral LMC neurons electroporated with either GFP only or [Unc5c]siRNA and GFP. Number of embryos quantified: n = 4 for all groups. (F) The expression of EphA4 and EphB1 is not altered after [Unc5]siRNA electroporation. (G) Detection of Isl1, Foxp1, GFP, and mouse Unc5c in chick embryos expressing mUnc5c and GFP. (H, I) The total number of LMC neurons and the proportion of medial and lateral LMC are normal after mUnc5c and GFP electroporation. Number of embryos quantified: n = 4 for all groups. (J) Equal proportion of medial and lateral LMC neurons electroporated with either GFP only or mUnc5c and GFP. Number of embryos quantified: n = 4 for all groups. (K) The expression of EphA4 and EphB1 is not altered after mUnc5c and GFP electroporation. Number of embryos quantified: n = 4 for all groups. (L) Detection of GFP, mUnc5c, mEphb2, and Foxp1 in chick embryos expressing normal (top) or low (1/5th) (bottom) concentrations of Ephb2-GFP and Unc5c plasmids. (M) Quantification of the ratio of the ectopic expression levels of low and normal concentration of GFP, Unc5c, and EphB2 in chick embryos. Number of embryos quantified: n = 6 for all groups. (N) Analysis of lateral LMC motor axon projections in chick embryos electroporated with Unc5c, Ephb2, Unc5c and Ephb2, or GFP expression plasmids (all at low concentrations). A retrograde tracer (HRP, blue) was injected in the ventral forelimb of HH st. 29/30 chick embryos followed by detection of Lhx1 (red) to identify lateral LMC neurons. Insets show examples of magnified HRP+ backfilled cells that are Lhx1+ (in low Unc5c + low Ephb2 co-electroporation) or Lhx1- (in all other conditions). (O) Quantification of retrogradely labeled lateral LMC axon projections. The graph depicts the mean percentage ± SD of electroporated (GFP+) and HRP+ backfilled motor neurons that express the lateral LMC marker Lhx1 after a ventral limb injection. N ≥ 3 embryos. HRP, horseradish peroxidase; LMC, lateral motor column; error bars = SD; *** = p<0.001; n.s. = not significant; statistical significance computed using Mann-Whitney U test (B–E, H–J, M), or Fisher’s exact test on raw numbers (O); all values are mean ± SD. Scale bars: (A, G, L) 56 μm; (F, K) 145 μm; (N) 40 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.011
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fig4s1: Characterization of siRNA-mediated Unc5c knockdown and Unc5c overexpression in LMC neurons in chicken.(A) Detection of Isl1, Foxp1, GFP, and Unc5c in chick embryos electroporated with [Unc5c]siRNA and GFP. (B) Quantification of the reduction in expression levels of Unc5c in LMC neurons after in ovo electroporation of [Unc5c]siRNA. Note a significant decrease (p<0.001) in Unc5c expression levels by 50% upon [Unc5c]siRNA and GFP but not GFP-only electroporation. Number of embryos quantified: n = 3 for both groups. (C, D) The total number of LMC neurons and the proportion of medial and lateral LMC are normal after [Unc5]siRNA electroporation. Number of embryos quantified: n = 4 for all groups. (E) Equal proportion of medial and lateral LMC neurons electroporated with either GFP only or [Unc5c]siRNA and GFP. Number of embryos quantified: n = 4 for all groups. (F) The expression of EphA4 and EphB1 is not altered after [Unc5]siRNA electroporation. (G) Detection of Isl1, Foxp1, GFP, and mouse Unc5c in chick embryos expressing mUnc5c and GFP. (H, I) The total number of LMC neurons and the proportion of medial and lateral LMC are normal after mUnc5c and GFP electroporation. Number of embryos quantified: n = 4 for all groups. (J) Equal proportion of medial and lateral LMC neurons electroporated with either GFP only or mUnc5c and GFP. Number of embryos quantified: n = 4 for all groups. (K) The expression of EphA4 and EphB1 is not altered after mUnc5c and GFP electroporation. Number of embryos quantified: n = 4 for all groups. (L) Detection of GFP, mUnc5c, mEphb2, and Foxp1 in chick embryos expressing normal (top) or low (1/5th) (bottom) concentrations of Ephb2-GFP and Unc5c plasmids. (M) Quantification of the ratio of the ectopic expression levels of low and normal concentration of GFP, Unc5c, and EphB2 in chick embryos. Number of embryos quantified: n = 6 for all groups. (N) Analysis of lateral LMC motor axon projections in chick embryos electroporated with Unc5c, Ephb2, Unc5c and Ephb2, or GFP expression plasmids (all at low concentrations). A retrograde tracer (HRP, blue) was injected in the ventral forelimb of HH st. 29/30 chick embryos followed by detection of Lhx1 (red) to identify lateral LMC neurons. Insets show examples of magnified HRP+ backfilled cells that are Lhx1+ (in low Unc5c + low Ephb2 co-electroporation) or Lhx1- (in all other conditions). (O) Quantification of retrogradely labeled lateral LMC axon projections. The graph depicts the mean percentage ± SD of electroporated (GFP+) and HRP+ backfilled motor neurons that express the lateral LMC marker Lhx1 after a ventral limb injection. N ≥ 3 embryos. HRP, horseradish peroxidase; LMC, lateral motor column; error bars = SD; *** = p<0.001; n.s. = not significant; statistical significance computed using Mann-Whitney U test (B–E, H–J, M), or Fisher’s exact test on raw numbers (O); all values are mean ± SD. Scale bars: (A, G, L) 56 μm; (F, K) 145 μm; (N) 40 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.011
Mentions: Given the prominent function of ephrins in motor axon guidance, we next investigated the possibility that Netrin and ephrin signals co-operate in LMC neurons. To do this we focused on the function of Unc5c and EphB2, an ephrin-B receptor guiding medial LMC axons in vivo (Luria et al., 2008). We first tested whether chicken Unc5c is required in vivo by reducing its expression through [Unc5c]siRNA and GFP plasmid electroporation of presumptive LMC neurons at HH st. 17–19. Analysis of the proportion of electroporated LMC axons entering the dorsal versus ventral limb nerves at HH st. 26 showed that in embryos electroporated with [Unc5c]siRNA, 72% of GFP+ axons were present in the dorsal limb nerve, compared with 52% in GFP electroporated controls (Figure 4A,B; Figure 4—figure supplement 1; p<0.01), demonstrating that chicken Unc5c is required for the fidelity of LMC trajectory choice.10.7554/eLife.10841.010Figure 4.Co-operation between Unc5c and EphB2 receptors in LMC trajectory selection. 

Bottom Line: We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors.Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals.Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Columbia University, New York, United States.

ABSTRACT
During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

No MeSH data available.


Related in: MedlinePlus