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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

Normal specification of LMC neurons in Unc5c, Dcc, and Ntn1Gt mutants and summary of LMC axon trajectory analysis in mutant mice.(A–C) Normal specification of LMC neurons in Unc5c-/-, Dcc-/-, and Ntn1Gt/Gt mice. (A) The number of medial and lateral LMC neurons was determined in e13.5 cervical and lumbar spinal cord sections from different mutant mice immunostained for Isl1 and Foxp1. (B) Quantification of medial and lateral LMC neurons in wt, Unc5-/-, Dcc-/-, and Ntn1Gt/Gt mice. (C) Normal expression of EphB1 and EphA4 in Unc5c-/-, Dcc-/-, and Ntn1Gt/Gt mice. E13.5 lumbar spinal cord sections from different mice were immunostained for Isl1 and EphA4 (C1-2, C5-6, C9-10) or analyzed by in situ hybridization for Isl1 and Ephb1 mRNA in consecutive sections (C3-4, C7-8, C11-12). (D and E) Summary of analysis of lateral (ventral labeling) and medial (dorsal labeling) LMC trajectories by backfill experiments in mice forelimbs of Neo1Gt/Gt double Dcc-/-;Neo1Gt/Gt, Unc5a-/-, double Unc5a-/-; Unc5c-/-, and Dscam-/- mice. Only the proportion of HRP+ LMC expressing Isl1 in dorsally filled double Unc5a-/-; Unc5c-/- embryos is significantly different from that of wt embryos (p<0.001). Number of embryos quantified for ventral fill: n = 5 (wt), 4 (Neo1Gt/Gt), 3 (Dcc-/- and Neo1Gt/Gt), 4 (Unc5a-/-), 4 (Unc5c-/- and Unc5a-/-), 4 (Dscam-/-). Number of embryos quantified for dorsal fill: n = 3 (wt), 4, (Neo1Gt/Gt), 4 (Dcc-/-; Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 3 (Dscam-/-). (F) Summary of analysis of lateral (ventral labeling) and medial (dorsal labeling) LMC trajectories by backfill experiments in e12.5 mice hindlimbs of different Netrin mutants as indicated. The proportions of HRP+ LMC expressing Isl1 in dorsally filled Unc5c-/-and Unc5a-/-; Unc5c-/- embryos are significantly different from that of wt embryos (p<0.001 for both groups). Number of embryos quantified for ventral fill: n = 7 (wt), 3, (Neo1Gt/Gt), 4 (Dcc-/-; Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 4 (Dscam-/-). Number of embryos quantified for dorsal fill: 4 (wt), 4, (Neo1Gt/Gt), 3 (Dcc-/- and Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 3 (Dscam-/-). HRP, horseradish peroxidase; LMC, lateral motor column; wt, wild-type; error bars = SD; *** = p<0.001; statistical significance computed using Student’s unpaired t test (B) or Fisher’s exact test on raw numbers (E, F); all values are mean ± SD. Quantification details in supplemental file 1C. Scale bars: (A) 50 μm; (C) 80 μm; (D) 40 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.007
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fig2s2: Normal specification of LMC neurons in Unc5c, Dcc, and Ntn1Gt mutants and summary of LMC axon trajectory analysis in mutant mice.(A–C) Normal specification of LMC neurons in Unc5c-/-, Dcc-/-, and Ntn1Gt/Gt mice. (A) The number of medial and lateral LMC neurons was determined in e13.5 cervical and lumbar spinal cord sections from different mutant mice immunostained for Isl1 and Foxp1. (B) Quantification of medial and lateral LMC neurons in wt, Unc5-/-, Dcc-/-, and Ntn1Gt/Gt mice. (C) Normal expression of EphB1 and EphA4 in Unc5c-/-, Dcc-/-, and Ntn1Gt/Gt mice. E13.5 lumbar spinal cord sections from different mice were immunostained for Isl1 and EphA4 (C1-2, C5-6, C9-10) or analyzed by in situ hybridization for Isl1 and Ephb1 mRNA in consecutive sections (C3-4, C7-8, C11-12). (D and E) Summary of analysis of lateral (ventral labeling) and medial (dorsal labeling) LMC trajectories by backfill experiments in mice forelimbs of Neo1Gt/Gt double Dcc-/-;Neo1Gt/Gt, Unc5a-/-, double Unc5a-/-; Unc5c-/-, and Dscam-/- mice. Only the proportion of HRP+ LMC expressing Isl1 in dorsally filled double Unc5a-/-; Unc5c-/- embryos is significantly different from that of wt embryos (p<0.001). Number of embryos quantified for ventral fill: n = 5 (wt), 4 (Neo1Gt/Gt), 3 (Dcc-/- and Neo1Gt/Gt), 4 (Unc5a-/-), 4 (Unc5c-/- and Unc5a-/-), 4 (Dscam-/-). Number of embryos quantified for dorsal fill: n = 3 (wt), 4, (Neo1Gt/Gt), 4 (Dcc-/-; Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 3 (Dscam-/-). (F) Summary of analysis of lateral (ventral labeling) and medial (dorsal labeling) LMC trajectories by backfill experiments in e12.5 mice hindlimbs of different Netrin mutants as indicated. The proportions of HRP+ LMC expressing Isl1 in dorsally filled Unc5c-/-and Unc5a-/-; Unc5c-/- embryos are significantly different from that of wt embryos (p<0.001 for both groups). Number of embryos quantified for ventral fill: n = 7 (wt), 3, (Neo1Gt/Gt), 4 (Dcc-/-; Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 4 (Dscam-/-). Number of embryos quantified for dorsal fill: 4 (wt), 4, (Neo1Gt/Gt), 3 (Dcc-/- and Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 3 (Dscam-/-). HRP, horseradish peroxidase; LMC, lateral motor column; wt, wild-type; error bars = SD; *** = p<0.001; statistical significance computed using Student’s unpaired t test (B) or Fisher’s exact test on raw numbers (E, F); all values are mean ± SD. Quantification details in supplemental file 1C. Scale bars: (A) 50 μm; (C) 80 μm; (D) 40 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.007

Mentions: We next determined whether loss of Netrin-1 or its receptors affects LMC axon pathfinding, by analyzing the limb trajectories of medial and lateral LMC axons in e11.5 and e12.5 wild-type, Ntn1Gt/Gt, Dcc-/-, Neo1Gt/Gt, Unc5c-/-, and Unc5a-/- mutant mice (Bae et al., 2009; Boyer and Kozak, 1991; Burgess et al., 2006; Fazeli et al., 1997; Williams et al., 2006). In all strains examined, the specification of LMC neurons, their axon outgrowth, and Eph and ephrin expression was normal (Figure 2—figure supplement 2; data not shown). To examine LMC axon trajectory selection, we injected the retrograde tracer horseradish peroxidase (HRP) into the ventral or dorsal limb muscles of Ntn1Gt/Gt and Netrin receptor mutant embryos and determined the divisional identity of labeled LMC neurons using the marker Foxp1 (Dasen et al., 2008; Rousso et al., 2008), and the medial LMC marker Isl1 (Luria et al., 2008). All our retrograde tracing observations were confirmed using an alkaline phosphatase medial LMC reporter transgene or Unc5c and EphA4 axonal staining (Figure 2—figure supplement 1). When HRP was injected into the ventral limb to detect aberrant lateral LMC axons, in Dcc mutants 9.7% of HRP-labeled neurons were medial LMC, compared with 4.4% in controls, 4.7% in Ntn1Gt/Gt and 5.6% in Unc5c-/- mice (Figure 2A–H; p=0.164, 1, and 0.748 for Dcc-/-, Ntn1Gt/Gt, and Unc5c-/- vs. controls; all values listed in Supplementary file 1B). We also injected HRP into the ventral limbs of Neo1Gt/Gt or Dscam-/- single or Dcc-/-; Neo1Gt/Gt double mutants and assessed the divisional identity of labeled LMC neurons (Figure 2—figure supplement 2). Neither single nor double mutant lateral LMC axons were found in the ventral limb at an incidence greater than that in control embryos (Figure 2—figure supplement 2; p=0.105, 0.537 and 0.537 for Neo1Gt/Gt, Dscam-/- and Dcc-/-; Neo1Gt/Gt vs. wild-type controls). Thus, extensive redundancy notwithstanding, Dcc, Neogenin, or Dscam are not required for in vivo lateral LMC axon guidance.10.7554/eLife.10841.005Figure 2.The requirement of Netrin-1 and its receptors for the fidelity of LMC axon 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)

Normal specification of LMC neurons in Unc5c, Dcc, and Ntn1Gt mutants and summary of LMC axon trajectory analysis in mutant mice.(A–C) Normal specification of LMC neurons in Unc5c-/-, Dcc-/-, and Ntn1Gt/Gt mice. (A) The number of medial and lateral LMC neurons was determined in e13.5 cervical and lumbar spinal cord sections from different mutant mice immunostained for Isl1 and Foxp1. (B) Quantification of medial and lateral LMC neurons in wt, Unc5-/-, Dcc-/-, and Ntn1Gt/Gt mice. (C) Normal expression of EphB1 and EphA4 in Unc5c-/-, Dcc-/-, and Ntn1Gt/Gt mice. E13.5 lumbar spinal cord sections from different mice were immunostained for Isl1 and EphA4 (C1-2, C5-6, C9-10) or analyzed by in situ hybridization for Isl1 and Ephb1 mRNA in consecutive sections (C3-4, C7-8, C11-12). (D and E) Summary of analysis of lateral (ventral labeling) and medial (dorsal labeling) LMC trajectories by backfill experiments in mice forelimbs of Neo1Gt/Gt double Dcc-/-;Neo1Gt/Gt, Unc5a-/-, double Unc5a-/-; Unc5c-/-, and Dscam-/- mice. Only the proportion of HRP+ LMC expressing Isl1 in dorsally filled double Unc5a-/-; Unc5c-/- embryos is significantly different from that of wt embryos (p<0.001). Number of embryos quantified for ventral fill: n = 5 (wt), 4 (Neo1Gt/Gt), 3 (Dcc-/- and Neo1Gt/Gt), 4 (Unc5a-/-), 4 (Unc5c-/- and Unc5a-/-), 4 (Dscam-/-). Number of embryos quantified for dorsal fill: n = 3 (wt), 4, (Neo1Gt/Gt), 4 (Dcc-/-; Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 3 (Dscam-/-). (F) Summary of analysis of lateral (ventral labeling) and medial (dorsal labeling) LMC trajectories by backfill experiments in e12.5 mice hindlimbs of different Netrin mutants as indicated. The proportions of HRP+ LMC expressing Isl1 in dorsally filled Unc5c-/-and Unc5a-/-; Unc5c-/- embryos are significantly different from that of wt embryos (p<0.001 for both groups). Number of embryos quantified for ventral fill: n = 7 (wt), 3, (Neo1Gt/Gt), 4 (Dcc-/-; Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 4 (Dscam-/-). Number of embryos quantified for dorsal fill: 4 (wt), 4, (Neo1Gt/Gt), 3 (Dcc-/- and Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 3 (Dscam-/-). HRP, horseradish peroxidase; LMC, lateral motor column; wt, wild-type; error bars = SD; *** = p<0.001; statistical significance computed using Student’s unpaired t test (B) or Fisher’s exact test on raw numbers (E, F); all values are mean ± SD. Quantification details in supplemental file 1C. Scale bars: (A) 50 μm; (C) 80 μm; (D) 40 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.007
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fig2s2: Normal specification of LMC neurons in Unc5c, Dcc, and Ntn1Gt mutants and summary of LMC axon trajectory analysis in mutant mice.(A–C) Normal specification of LMC neurons in Unc5c-/-, Dcc-/-, and Ntn1Gt/Gt mice. (A) The number of medial and lateral LMC neurons was determined in e13.5 cervical and lumbar spinal cord sections from different mutant mice immunostained for Isl1 and Foxp1. (B) Quantification of medial and lateral LMC neurons in wt, Unc5-/-, Dcc-/-, and Ntn1Gt/Gt mice. (C) Normal expression of EphB1 and EphA4 in Unc5c-/-, Dcc-/-, and Ntn1Gt/Gt mice. E13.5 lumbar spinal cord sections from different mice were immunostained for Isl1 and EphA4 (C1-2, C5-6, C9-10) or analyzed by in situ hybridization for Isl1 and Ephb1 mRNA in consecutive sections (C3-4, C7-8, C11-12). (D and E) Summary of analysis of lateral (ventral labeling) and medial (dorsal labeling) LMC trajectories by backfill experiments in mice forelimbs of Neo1Gt/Gt double Dcc-/-;Neo1Gt/Gt, Unc5a-/-, double Unc5a-/-; Unc5c-/-, and Dscam-/- mice. Only the proportion of HRP+ LMC expressing Isl1 in dorsally filled double Unc5a-/-; Unc5c-/- embryos is significantly different from that of wt embryos (p<0.001). Number of embryos quantified for ventral fill: n = 5 (wt), 4 (Neo1Gt/Gt), 3 (Dcc-/- and Neo1Gt/Gt), 4 (Unc5a-/-), 4 (Unc5c-/- and Unc5a-/-), 4 (Dscam-/-). Number of embryos quantified for dorsal fill: n = 3 (wt), 4, (Neo1Gt/Gt), 4 (Dcc-/-; Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 3 (Dscam-/-). (F) Summary of analysis of lateral (ventral labeling) and medial (dorsal labeling) LMC trajectories by backfill experiments in e12.5 mice hindlimbs of different Netrin mutants as indicated. The proportions of HRP+ LMC expressing Isl1 in dorsally filled Unc5c-/-and Unc5a-/-; Unc5c-/- embryos are significantly different from that of wt embryos (p<0.001 for both groups). Number of embryos quantified for ventral fill: n = 7 (wt), 3, (Neo1Gt/Gt), 4 (Dcc-/-; Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 4 (Dscam-/-). Number of embryos quantified for dorsal fill: 4 (wt), 4, (Neo1Gt/Gt), 3 (Dcc-/- and Neo1Gt/Gt), 4 (Unc5a-/-), 3 (Unc5c-/-; Unc5a-/-), 3 (Dscam-/-). HRP, horseradish peroxidase; LMC, lateral motor column; wt, wild-type; error bars = SD; *** = p<0.001; statistical significance computed using Student’s unpaired t test (B) or Fisher’s exact test on raw numbers (E, F); all values are mean ± SD. Quantification details in supplemental file 1C. Scale bars: (A) 50 μm; (C) 80 μm; (D) 40 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.007
Mentions: We next determined whether loss of Netrin-1 or its receptors affects LMC axon pathfinding, by analyzing the limb trajectories of medial and lateral LMC axons in e11.5 and e12.5 wild-type, Ntn1Gt/Gt, Dcc-/-, Neo1Gt/Gt, Unc5c-/-, and Unc5a-/- mutant mice (Bae et al., 2009; Boyer and Kozak, 1991; Burgess et al., 2006; Fazeli et al., 1997; Williams et al., 2006). In all strains examined, the specification of LMC neurons, their axon outgrowth, and Eph and ephrin expression was normal (Figure 2—figure supplement 2; data not shown). To examine LMC axon trajectory selection, we injected the retrograde tracer horseradish peroxidase (HRP) into the ventral or dorsal limb muscles of Ntn1Gt/Gt and Netrin receptor mutant embryos and determined the divisional identity of labeled LMC neurons using the marker Foxp1 (Dasen et al., 2008; Rousso et al., 2008), and the medial LMC marker Isl1 (Luria et al., 2008). All our retrograde tracing observations were confirmed using an alkaline phosphatase medial LMC reporter transgene or Unc5c and EphA4 axonal staining (Figure 2—figure supplement 1). When HRP was injected into the ventral limb to detect aberrant lateral LMC axons, in Dcc mutants 9.7% of HRP-labeled neurons were medial LMC, compared with 4.4% in controls, 4.7% in Ntn1Gt/Gt and 5.6% in Unc5c-/- mice (Figure 2A–H; p=0.164, 1, and 0.748 for Dcc-/-, Ntn1Gt/Gt, and Unc5c-/- vs. controls; all values listed in Supplementary file 1B). We also injected HRP into the ventral limbs of Neo1Gt/Gt or Dscam-/- single or Dcc-/-; Neo1Gt/Gt double mutants and assessed the divisional identity of labeled LMC neurons (Figure 2—figure supplement 2). Neither single nor double mutant lateral LMC axons were found in the ventral limb at an incidence greater than that in control embryos (Figure 2—figure supplement 2; p=0.105, 0.537 and 0.537 for Neo1Gt/Gt, Dscam-/- and Dcc-/-; Neo1Gt/Gt vs. wild-type controls). Thus, extensive redundancy notwithstanding, Dcc, Neogenin, or Dscam are not required for in vivo lateral LMC axon guidance.10.7554/eLife.10841.005Figure 2.The requirement of Netrin-1 and its receptors for the fidelity of LMC axon 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