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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.

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Related in: MedlinePlus

Unc5c–EphB2 receptor association.(A) Unc5c and EphB2 protein localization in permeabilized LMC growth cones treated for 15 min with Fc, Netrin-1, eB2, Netrin-1+eB2. Individual channels are inverted. (B) Growth cone size does not change following Fc, eB2, Netrin-1, or Netrin-1+eB2 treatment for 15 min (p=0.8161 using one-way ANOVA; N = 3; n ≥ 36 growth cones per treatment). All treatments result in same receptor protein signal levels. (C) Quantification of Unc5c protein signal in LMC growth cones as fraction of growth cone area above threshold (p=0.3643, one-way ANOVA and Tukey's multiple comparisons test; N = 3; n ≥ 36 growth cones per treatment). (D) Quantification of EphB2 protein signal in LMC growth cones as fraction of growth cone area above threshold (p=0.3862, one-way ANOVA and Tukey's multiple comparisons test; N = 3; n ≥ 36 growth cones per treatment). (E) Unc5c and EphB2 protein localization in non-permeabilized LMC growth cones treated with eB2 or Netrin-1 for 15 min. Individual channels are inverted. Receptor clusters are depicted in insets, arrowheads: Unc5c and EphB2 co-localization. (F) Costes’ P-value analysis for Fc-treated, eB2-treated, Netrin-1-treated, and Netrin-1+eB2-treated non-permeabilized growth cones, calculated by automatically shuffling appropriately sized chunks of one of the channels of an image and running co-localization analysis. This was done 100 times per image. A value of 1 signifies that 100% of the shuffled results had a Pearson's R value lower than the one calculated for the original image, i.e. observed co-localization is higher than expected by chance (N = 3; n ≥ 33 growth cones per treatment). (G) Quantification of growth cone size for non-permeabilized growth experiments (p=0.1675 using one-way ANOVA, N = 3; n ≥ 33 growth cones per treatment). (H) Quantification of Unc5c protein signal in LMC growth cones as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment; detailed values in supplemental file 1C). (I) Quantification of EphB2 protein signal in LMC growth cones as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment). (J) Quantification of phalloidin signal reflecting successful non-permeabilized staining, expressed as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment). (K) Permeabilized versus non-permeabilized signal for fluorescent-conjugated phalloidin and early endosome marker 1 (EEA1), showing successful staining with minimal membrane permeabilization. n.s. = not significant; error bars = SD. (L) Specificity of antibodies in immunoblot detection. HEK293 cells were transfected with Unc5c-Myc or EphB2-GFP and anti-Myc and anti-GFP antibodies were used in western blots to detect expressed proteins from total lysates. (M) Co-immunoprecipitation of EphB2 by Unc5c. EphB2-GFP was immunoprecipitated by Myc antibody from lysates of Unc5c-Myc-transfected and Ephb2-GFP-transfected HEK293 cells. (N) Co-immunoprecipitated Unc5c fold changes. Pixel intensity and area of unsaturated western blot bands were measured in inverted images in Photoshop and total intensity calculated. For fold changes calculations, values of co-immunoprecipitated Unc5c in each treatment were normalized to immunoprecipitated EphB2-GFP and compared with Fc condition. One sample t-test, * = p<0.05; N=4 in each condition. (O) Reduction in co-immunoprecipitated Unc5c by EphB2-KD-GFP when compared with EphB2-GFP interactions. Calculation of band intensities were done as described in (N). Values of co-immunoprecipitated Unc5c-Myc were normalized to immunoprecipitated EphB2-GFP or EphB2-KD-GFP and the fold change between EphB2-KD-GFP and EphB2-GFP calculated. One sample t-test, * = p<0.05; N=4 in each condition. LMC, lateral motor column; scale bars: (A, E, K) 2 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.015
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fig6s1: Unc5c–EphB2 receptor association.(A) Unc5c and EphB2 protein localization in permeabilized LMC growth cones treated for 15 min with Fc, Netrin-1, eB2, Netrin-1+eB2. Individual channels are inverted. (B) Growth cone size does not change following Fc, eB2, Netrin-1, or Netrin-1+eB2 treatment for 15 min (p=0.8161 using one-way ANOVA; N = 3; n ≥ 36 growth cones per treatment). All treatments result in same receptor protein signal levels. (C) Quantification of Unc5c protein signal in LMC growth cones as fraction of growth cone area above threshold (p=0.3643, one-way ANOVA and Tukey's multiple comparisons test; N = 3; n ≥ 36 growth cones per treatment). (D) Quantification of EphB2 protein signal in LMC growth cones as fraction of growth cone area above threshold (p=0.3862, one-way ANOVA and Tukey's multiple comparisons test; N = 3; n ≥ 36 growth cones per treatment). (E) Unc5c and EphB2 protein localization in non-permeabilized LMC growth cones treated with eB2 or Netrin-1 for 15 min. Individual channels are inverted. Receptor clusters are depicted in insets, arrowheads: Unc5c and EphB2 co-localization. (F) Costes’ P-value analysis for Fc-treated, eB2-treated, Netrin-1-treated, and Netrin-1+eB2-treated non-permeabilized growth cones, calculated by automatically shuffling appropriately sized chunks of one of the channels of an image and running co-localization analysis. This was done 100 times per image. A value of 1 signifies that 100% of the shuffled results had a Pearson's R value lower than the one calculated for the original image, i.e. observed co-localization is higher than expected by chance (N = 3; n ≥ 33 growth cones per treatment). (G) Quantification of growth cone size for non-permeabilized growth experiments (p=0.1675 using one-way ANOVA, N = 3; n ≥ 33 growth cones per treatment). (H) Quantification of Unc5c protein signal in LMC growth cones as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment; detailed values in supplemental file 1C). (I) Quantification of EphB2 protein signal in LMC growth cones as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment). (J) Quantification of phalloidin signal reflecting successful non-permeabilized staining, expressed as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment). (K) Permeabilized versus non-permeabilized signal for fluorescent-conjugated phalloidin and early endosome marker 1 (EEA1), showing successful staining with minimal membrane permeabilization. n.s. = not significant; error bars = SD. (L) Specificity of antibodies in immunoblot detection. HEK293 cells were transfected with Unc5c-Myc or EphB2-GFP and anti-Myc and anti-GFP antibodies were used in western blots to detect expressed proteins from total lysates. (M) Co-immunoprecipitation of EphB2 by Unc5c. EphB2-GFP was immunoprecipitated by Myc antibody from lysates of Unc5c-Myc-transfected and Ephb2-GFP-transfected HEK293 cells. (N) Co-immunoprecipitated Unc5c fold changes. Pixel intensity and area of unsaturated western blot bands were measured in inverted images in Photoshop and total intensity calculated. For fold changes calculations, values of co-immunoprecipitated Unc5c in each treatment were normalized to immunoprecipitated EphB2-GFP and compared with Fc condition. One sample t-test, * = p<0.05; N=4 in each condition. (O) Reduction in co-immunoprecipitated Unc5c by EphB2-KD-GFP when compared with EphB2-GFP interactions. Calculation of band intensities were done as described in (N). Values of co-immunoprecipitated Unc5c-Myc were normalized to immunoprecipitated EphB2-GFP or EphB2-KD-GFP and the fold change between EphB2-KD-GFP and EphB2-GFP calculated. One sample t-test, * = p<0.05; N=4 in each condition. LMC, lateral motor column; scale bars: (A, E, K) 2 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.015

Mentions: (A) Medial LMC neuron explant growth cone collapse assay scheme. (B) Percentage of collapsed e[Isl1]::GFP medial LMC growth cones following 30 min exposure to Fc (10 μg/ml), eB2-Fc (high: 10 μg/ml; low: 1 μg/ml), Netrin-1 (300 ng/ml) or Netrin-1 and eB2-Fc (300 ng/ml and 1 μg/ml). Significance computed using Fisher’s exact test. (C) Examples of growth cones labeled with Tuj1 (green) and phalloidin (red). (D–I) Unc5c and EphB2 protein localization in non-permeabilized LMC growth cones treated with Fc or eB2 and Netrin-1 for 15 min. Individual channels are inverted. All treatments result in same receptor protein signal levels (eB2 and Netrin-1 images are in Figure 6—figure supplement 1). Receptor clusters are depicted in insets, arrowheads: Unc5c and EphB2 co-localization. (J) Pearson's R value as a measure of surface Unc5c and EphB2 co-localization in LMC growth cones. Co-localization levels are higher than expected by chance, as demonstrated by Costes’ shuffled image P-value calculations (Figure 6—figure supplement 1). Ligand treatment does not increase the levels of receptor co-localization observed (p=0.7940, one-way analysis of variance (ANOVA) and Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment). (K) Unc5c and EphB2 receptor interactions. Unc5c-Myc was co-immunoprecipitated with EphB2-GFP but not with EphA3-GFP in transfected HEK-293 cells. All samples shown were run in same gel. (L) Unc5c-EphB2 interaction is selectively enhanced by 15 min incubation with eB2-Fc (1.5 μg/ml) or eB2-Fc+Netrin-1 (1.5 μg/ml +250ng/ml) but not with Netrin-1 (250 ng/ml), Fc (1.5 μg/ml), or ephrin-A3-Fc (1.5 μg/ml) prior to lysates preparation. Fc fusion proteins were pre-clustered by incubating them with anti-human or anti-mouse Ig for 1 hr at 4°C. For quantifications see Figure 6—figure supplement 1N. (M) Comparison of Unc5c interactions with wild-type or kinase-dead EphB2. Unc5c-Myc/EphB2-GFP interaction is blocked when a single point mutation is introduced in EphB2-GFP abolishing its kinase function (EphB2-KD-GFP, Dalva et al., 2000). For quantifications see Figure 6—figure supplement 1O. All samples shown were run in same gel. (N) P-EphB2 levels are increased upon stimulation with Netrin-1+eB2-Fc compared with eB2-Fc alone. p-EphB2 was developed first, followed by stripping of the membrane and re-blotting with anti-EphB2 antibody. Two replicate comparisons are shown; one sample t-test; p<0.02, N=10 comparisons, 4 experiments. eB2, ephrin-B2-Fc; ip, immunoprecipitation; LMC, lateral motor column; tr, transfection. All error bars = SD; *** = p<0.001; n.s. = not significant; scale bars: (C) 10 μm; (D–I) 2 μm. All values (mean ± SD) can be found in Supplementary file 1B.


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)

Unc5c–EphB2 receptor association.(A) Unc5c and EphB2 protein localization in permeabilized LMC growth cones treated for 15 min with Fc, Netrin-1, eB2, Netrin-1+eB2. Individual channels are inverted. (B) Growth cone size does not change following Fc, eB2, Netrin-1, or Netrin-1+eB2 treatment for 15 min (p=0.8161 using one-way ANOVA; N = 3; n ≥ 36 growth cones per treatment). All treatments result in same receptor protein signal levels. (C) Quantification of Unc5c protein signal in LMC growth cones as fraction of growth cone area above threshold (p=0.3643, one-way ANOVA and Tukey's multiple comparisons test; N = 3; n ≥ 36 growth cones per treatment). (D) Quantification of EphB2 protein signal in LMC growth cones as fraction of growth cone area above threshold (p=0.3862, one-way ANOVA and Tukey's multiple comparisons test; N = 3; n ≥ 36 growth cones per treatment). (E) Unc5c and EphB2 protein localization in non-permeabilized LMC growth cones treated with eB2 or Netrin-1 for 15 min. Individual channels are inverted. Receptor clusters are depicted in insets, arrowheads: Unc5c and EphB2 co-localization. (F) Costes’ P-value analysis for Fc-treated, eB2-treated, Netrin-1-treated, and Netrin-1+eB2-treated non-permeabilized growth cones, calculated by automatically shuffling appropriately sized chunks of one of the channels of an image and running co-localization analysis. This was done 100 times per image. A value of 1 signifies that 100% of the shuffled results had a Pearson's R value lower than the one calculated for the original image, i.e. observed co-localization is higher than expected by chance (N = 3; n ≥ 33 growth cones per treatment). (G) Quantification of growth cone size for non-permeabilized growth experiments (p=0.1675 using one-way ANOVA, N = 3; n ≥ 33 growth cones per treatment). (H) Quantification of Unc5c protein signal in LMC growth cones as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment; detailed values in supplemental file 1C). (I) Quantification of EphB2 protein signal in LMC growth cones as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment). (J) Quantification of phalloidin signal reflecting successful non-permeabilized staining, expressed as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment). (K) Permeabilized versus non-permeabilized signal for fluorescent-conjugated phalloidin and early endosome marker 1 (EEA1), showing successful staining with minimal membrane permeabilization. n.s. = not significant; error bars = SD. (L) Specificity of antibodies in immunoblot detection. HEK293 cells were transfected with Unc5c-Myc or EphB2-GFP and anti-Myc and anti-GFP antibodies were used in western blots to detect expressed proteins from total lysates. (M) Co-immunoprecipitation of EphB2 by Unc5c. EphB2-GFP was immunoprecipitated by Myc antibody from lysates of Unc5c-Myc-transfected and Ephb2-GFP-transfected HEK293 cells. (N) Co-immunoprecipitated Unc5c fold changes. Pixel intensity and area of unsaturated western blot bands were measured in inverted images in Photoshop and total intensity calculated. For fold changes calculations, values of co-immunoprecipitated Unc5c in each treatment were normalized to immunoprecipitated EphB2-GFP and compared with Fc condition. One sample t-test, * = p<0.05; N=4 in each condition. (O) Reduction in co-immunoprecipitated Unc5c by EphB2-KD-GFP when compared with EphB2-GFP interactions. Calculation of band intensities were done as described in (N). Values of co-immunoprecipitated Unc5c-Myc were normalized to immunoprecipitated EphB2-GFP or EphB2-KD-GFP and the fold change between EphB2-KD-GFP and EphB2-GFP calculated. One sample t-test, * = p<0.05; N=4 in each condition. LMC, lateral motor column; scale bars: (A, E, K) 2 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.015
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fig6s1: Unc5c–EphB2 receptor association.(A) Unc5c and EphB2 protein localization in permeabilized LMC growth cones treated for 15 min with Fc, Netrin-1, eB2, Netrin-1+eB2. Individual channels are inverted. (B) Growth cone size does not change following Fc, eB2, Netrin-1, or Netrin-1+eB2 treatment for 15 min (p=0.8161 using one-way ANOVA; N = 3; n ≥ 36 growth cones per treatment). All treatments result in same receptor protein signal levels. (C) Quantification of Unc5c protein signal in LMC growth cones as fraction of growth cone area above threshold (p=0.3643, one-way ANOVA and Tukey's multiple comparisons test; N = 3; n ≥ 36 growth cones per treatment). (D) Quantification of EphB2 protein signal in LMC growth cones as fraction of growth cone area above threshold (p=0.3862, one-way ANOVA and Tukey's multiple comparisons test; N = 3; n ≥ 36 growth cones per treatment). (E) Unc5c and EphB2 protein localization in non-permeabilized LMC growth cones treated with eB2 or Netrin-1 for 15 min. Individual channels are inverted. Receptor clusters are depicted in insets, arrowheads: Unc5c and EphB2 co-localization. (F) Costes’ P-value analysis for Fc-treated, eB2-treated, Netrin-1-treated, and Netrin-1+eB2-treated non-permeabilized growth cones, calculated by automatically shuffling appropriately sized chunks of one of the channels of an image and running co-localization analysis. This was done 100 times per image. A value of 1 signifies that 100% of the shuffled results had a Pearson's R value lower than the one calculated for the original image, i.e. observed co-localization is higher than expected by chance (N = 3; n ≥ 33 growth cones per treatment). (G) Quantification of growth cone size for non-permeabilized growth experiments (p=0.1675 using one-way ANOVA, N = 3; n ≥ 33 growth cones per treatment). (H) Quantification of Unc5c protein signal in LMC growth cones as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment; detailed values in supplemental file 1C). (I) Quantification of EphB2 protein signal in LMC growth cones as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment). (J) Quantification of phalloidin signal reflecting successful non-permeabilized staining, expressed as fraction of growth cone area above threshold (no significant differences, except between permeabilized and non-permeabilized, p<0.05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment). (K) Permeabilized versus non-permeabilized signal for fluorescent-conjugated phalloidin and early endosome marker 1 (EEA1), showing successful staining with minimal membrane permeabilization. n.s. = not significant; error bars = SD. (L) Specificity of antibodies in immunoblot detection. HEK293 cells were transfected with Unc5c-Myc or EphB2-GFP and anti-Myc and anti-GFP antibodies were used in western blots to detect expressed proteins from total lysates. (M) Co-immunoprecipitation of EphB2 by Unc5c. EphB2-GFP was immunoprecipitated by Myc antibody from lysates of Unc5c-Myc-transfected and Ephb2-GFP-transfected HEK293 cells. (N) Co-immunoprecipitated Unc5c fold changes. Pixel intensity and area of unsaturated western blot bands were measured in inverted images in Photoshop and total intensity calculated. For fold changes calculations, values of co-immunoprecipitated Unc5c in each treatment were normalized to immunoprecipitated EphB2-GFP and compared with Fc condition. One sample t-test, * = p<0.05; N=4 in each condition. (O) Reduction in co-immunoprecipitated Unc5c by EphB2-KD-GFP when compared with EphB2-GFP interactions. Calculation of band intensities were done as described in (N). Values of co-immunoprecipitated Unc5c-Myc were normalized to immunoprecipitated EphB2-GFP or EphB2-KD-GFP and the fold change between EphB2-KD-GFP and EphB2-GFP calculated. One sample t-test, * = p<0.05; N=4 in each condition. LMC, lateral motor column; scale bars: (A, E, K) 2 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.015
Mentions: (A) Medial LMC neuron explant growth cone collapse assay scheme. (B) Percentage of collapsed e[Isl1]::GFP medial LMC growth cones following 30 min exposure to Fc (10 μg/ml), eB2-Fc (high: 10 μg/ml; low: 1 μg/ml), Netrin-1 (300 ng/ml) or Netrin-1 and eB2-Fc (300 ng/ml and 1 μg/ml). Significance computed using Fisher’s exact test. (C) Examples of growth cones labeled with Tuj1 (green) and phalloidin (red). (D–I) Unc5c and EphB2 protein localization in non-permeabilized LMC growth cones treated with Fc or eB2 and Netrin-1 for 15 min. Individual channels are inverted. All treatments result in same receptor protein signal levels (eB2 and Netrin-1 images are in Figure 6—figure supplement 1). Receptor clusters are depicted in insets, arrowheads: Unc5c and EphB2 co-localization. (J) Pearson's R value as a measure of surface Unc5c and EphB2 co-localization in LMC growth cones. Co-localization levels are higher than expected by chance, as demonstrated by Costes’ shuffled image P-value calculations (Figure 6—figure supplement 1). Ligand treatment does not increase the levels of receptor co-localization observed (p=0.7940, one-way analysis of variance (ANOVA) and Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment). (K) Unc5c and EphB2 receptor interactions. Unc5c-Myc was co-immunoprecipitated with EphB2-GFP but not with EphA3-GFP in transfected HEK-293 cells. All samples shown were run in same gel. (L) Unc5c-EphB2 interaction is selectively enhanced by 15 min incubation with eB2-Fc (1.5 μg/ml) or eB2-Fc+Netrin-1 (1.5 μg/ml +250ng/ml) but not with Netrin-1 (250 ng/ml), Fc (1.5 μg/ml), or ephrin-A3-Fc (1.5 μg/ml) prior to lysates preparation. Fc fusion proteins were pre-clustered by incubating them with anti-human or anti-mouse Ig for 1 hr at 4°C. For quantifications see Figure 6—figure supplement 1N. (M) Comparison of Unc5c interactions with wild-type or kinase-dead EphB2. Unc5c-Myc/EphB2-GFP interaction is blocked when a single point mutation is introduced in EphB2-GFP abolishing its kinase function (EphB2-KD-GFP, Dalva et al., 2000). For quantifications see Figure 6—figure supplement 1O. All samples shown were run in same gel. (N) P-EphB2 levels are increased upon stimulation with Netrin-1+eB2-Fc compared with eB2-Fc alone. p-EphB2 was developed first, followed by stripping of the membrane and re-blotting with anti-EphB2 antibody. Two replicate comparisons are shown; one sample t-test; p<0.02, N=10 comparisons, 4 experiments. eB2, ephrin-B2-Fc; ip, immunoprecipitation; LMC, lateral motor column; tr, transfection. All error bars = SD; *** = p<0.001; n.s. = not significant; scale bars: (C) 10 μm; (D–I) 2 μm. All values (mean ± SD) can be found in Supplementary file 1B.

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