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Integration of shallow gradients of Shh and Netrin-1 guides commissural axons.

Sloan TF, Qasaimeh MA, Juncker D, Yam PT, Charron F - PLoS Biol. (2015)

Bottom Line: We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow.We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1.Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada; Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada; Program in Neuroengineering, McGill University, Montreal, Quebec, Canada.

ABSTRACT
During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.

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Axons turn up gradients of Shh and Netrin-1 in le Massif.(A) The turned angle is defined as the angle between the line representing the proximal 20 μm of the axon at the cell body (axon base, turquoise) and the line representing the distal 20 μm of the axon at the growth cone (axon tip, red). The sign of the turned angle is positive if the turn is in the direction of the higher concentration of chemoattractant, and the sign of the turned angle is negative if the turn is in the direction of the lower concentration. (B,D) Images of commissural neurons grown for 1 d in culture followed by the application of (B) control vehicle gradient (BSA), (C) Shh gradient or (D) Netrin-1 gradient for 24 h. Wedge represents the gradient orientation. The average turned angles of axons as a function of the absolute concentration of (E) Shh and (F) Netrin-1 at the growth cone. Axons turn to a similar extent over a wide range of concentrations. The number of axons in each group is indicated in parentheses. (G,H) Circular distribution of individual turned angles. The angular deviation from vertical represents the magnitude of the turned angle, such that points to the right of the center are attracted (green) and those to the left are repelled (red). Neurons which turned between -5° and 5° are considered to be neutral (yellow). The distance of each point from the center represents the axon length. A random sample of 60 axons for Shh and Netrin-1 are plotted. A small shift in distribution towards attraction is seen across a wide range of concentrations for either cue. (I,J) To exclude the possibility that the gradients influence the angle at which the axon protrudes from the cell body, a gradient was applied 6 h following plating the neurons, before most neurons had initiated an axon. Axon base angle frequency distributions for axons grown in (I) Shh and (J) Netrin-1 were measured. Green bars represent the number of axons with a base angle facing up-gradient, and red bars indicate axons with angles facing down-gradient. There is no significant bias in angle distribution for either cue (Rayleigh test for uniformity, Shh: n = 2,028; Netrin: n = 2,805). Scale bars (B-D): 10 μm. (G,H): 25 μm. Error bars represent SEM. cb, cell body; gc, growth cone. See also S3 Fig.
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pbio.1002119.g003: Axons turn up gradients of Shh and Netrin-1 in le Massif.(A) The turned angle is defined as the angle between the line representing the proximal 20 μm of the axon at the cell body (axon base, turquoise) and the line representing the distal 20 μm of the axon at the growth cone (axon tip, red). The sign of the turned angle is positive if the turn is in the direction of the higher concentration of chemoattractant, and the sign of the turned angle is negative if the turn is in the direction of the lower concentration. (B,D) Images of commissural neurons grown for 1 d in culture followed by the application of (B) control vehicle gradient (BSA), (C) Shh gradient or (D) Netrin-1 gradient for 24 h. Wedge represents the gradient orientation. The average turned angles of axons as a function of the absolute concentration of (E) Shh and (F) Netrin-1 at the growth cone. Axons turn to a similar extent over a wide range of concentrations. The number of axons in each group is indicated in parentheses. (G,H) Circular distribution of individual turned angles. The angular deviation from vertical represents the magnitude of the turned angle, such that points to the right of the center are attracted (green) and those to the left are repelled (red). Neurons which turned between -5° and 5° are considered to be neutral (yellow). The distance of each point from the center represents the axon length. A random sample of 60 axons for Shh and Netrin-1 are plotted. A small shift in distribution towards attraction is seen across a wide range of concentrations for either cue. (I,J) To exclude the possibility that the gradients influence the angle at which the axon protrudes from the cell body, a gradient was applied 6 h following plating the neurons, before most neurons had initiated an axon. Axon base angle frequency distributions for axons grown in (I) Shh and (J) Netrin-1 were measured. Green bars represent the number of axons with a base angle facing up-gradient, and red bars indicate axons with angles facing down-gradient. There is no significant bias in angle distribution for either cue (Rayleigh test for uniformity, Shh: n = 2,028; Netrin: n = 2,805). Scale bars (B-D): 10 μm. (G,H): 25 μm. Error bars represent SEM. cb, cell body; gc, growth cone. See also S3 Fig.

Mentions: We established gradients of Shh or Netrin-1 after commissural neurons had been cultured for 24 h, when the majority of neurons had already initiated an axon. We calculated the turned angle of an axon as the difference between the base and tip angles (Fig. 3A) and scored the angle as positive if the axon turned towards the gradient and negative if it turned away. By varying the maximal concentration of ligand in a particular chamber, we could test a wide range of concentrations. In a control gradient (Phosphate buffered saline/BSA), we observed a wide range of turned angles towards and away from the gradient, resulting in a net turned angle of 0° (Fig. 3B,E). Neurons exposed to a gradient of Shh (Fig. 3C,E) or Netrin-1 (Fig. 3D,F), however, turned towards the higher concentration of chemoattractant. For either cue, we observed axon turning in response to a wide range of concentrations at the growth cone (Fig. 3E,F). The distribution of turned angles of individual axons confirmed that wide concentrations of Shh and Netrin-1 induced biases towards attraction (Fig. 3G,H). To eliminate the possibility that Shh or Netrin-1 influences the orientation at which the axon exits the cell body (axon base angle), thus confounding our measurement of the angle turned, we performed experiments where gradients were established 4–6 h after the neurons were plated, before the majority of neurons had initiated an axon. We found that Shh and Netrin-1 gradients induced no significant bias in the distribution of axon base angles facing up-gradient (higher concentration) compared to those facing down-gradient (lower concentration) (Fig. 3I,J). Therefore, le Massif generates gradients that can induce axon turning without any effect on axonal initiation.


Integration of shallow gradients of Shh and Netrin-1 guides commissural axons.

Sloan TF, Qasaimeh MA, Juncker D, Yam PT, Charron F - PLoS Biol. (2015)

Axons turn up gradients of Shh and Netrin-1 in le Massif.(A) The turned angle is defined as the angle between the line representing the proximal 20 μm of the axon at the cell body (axon base, turquoise) and the line representing the distal 20 μm of the axon at the growth cone (axon tip, red). The sign of the turned angle is positive if the turn is in the direction of the higher concentration of chemoattractant, and the sign of the turned angle is negative if the turn is in the direction of the lower concentration. (B,D) Images of commissural neurons grown for 1 d in culture followed by the application of (B) control vehicle gradient (BSA), (C) Shh gradient or (D) Netrin-1 gradient for 24 h. Wedge represents the gradient orientation. The average turned angles of axons as a function of the absolute concentration of (E) Shh and (F) Netrin-1 at the growth cone. Axons turn to a similar extent over a wide range of concentrations. The number of axons in each group is indicated in parentheses. (G,H) Circular distribution of individual turned angles. The angular deviation from vertical represents the magnitude of the turned angle, such that points to the right of the center are attracted (green) and those to the left are repelled (red). Neurons which turned between -5° and 5° are considered to be neutral (yellow). The distance of each point from the center represents the axon length. A random sample of 60 axons for Shh and Netrin-1 are plotted. A small shift in distribution towards attraction is seen across a wide range of concentrations for either cue. (I,J) To exclude the possibility that the gradients influence the angle at which the axon protrudes from the cell body, a gradient was applied 6 h following plating the neurons, before most neurons had initiated an axon. Axon base angle frequency distributions for axons grown in (I) Shh and (J) Netrin-1 were measured. Green bars represent the number of axons with a base angle facing up-gradient, and red bars indicate axons with angles facing down-gradient. There is no significant bias in angle distribution for either cue (Rayleigh test for uniformity, Shh: n = 2,028; Netrin: n = 2,805). Scale bars (B-D): 10 μm. (G,H): 25 μm. Error bars represent SEM. cb, cell body; gc, growth cone. See also S3 Fig.
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pbio.1002119.g003: Axons turn up gradients of Shh and Netrin-1 in le Massif.(A) The turned angle is defined as the angle between the line representing the proximal 20 μm of the axon at the cell body (axon base, turquoise) and the line representing the distal 20 μm of the axon at the growth cone (axon tip, red). The sign of the turned angle is positive if the turn is in the direction of the higher concentration of chemoattractant, and the sign of the turned angle is negative if the turn is in the direction of the lower concentration. (B,D) Images of commissural neurons grown for 1 d in culture followed by the application of (B) control vehicle gradient (BSA), (C) Shh gradient or (D) Netrin-1 gradient for 24 h. Wedge represents the gradient orientation. The average turned angles of axons as a function of the absolute concentration of (E) Shh and (F) Netrin-1 at the growth cone. Axons turn to a similar extent over a wide range of concentrations. The number of axons in each group is indicated in parentheses. (G,H) Circular distribution of individual turned angles. The angular deviation from vertical represents the magnitude of the turned angle, such that points to the right of the center are attracted (green) and those to the left are repelled (red). Neurons which turned between -5° and 5° are considered to be neutral (yellow). The distance of each point from the center represents the axon length. A random sample of 60 axons for Shh and Netrin-1 are plotted. A small shift in distribution towards attraction is seen across a wide range of concentrations for either cue. (I,J) To exclude the possibility that the gradients influence the angle at which the axon protrudes from the cell body, a gradient was applied 6 h following plating the neurons, before most neurons had initiated an axon. Axon base angle frequency distributions for axons grown in (I) Shh and (J) Netrin-1 were measured. Green bars represent the number of axons with a base angle facing up-gradient, and red bars indicate axons with angles facing down-gradient. There is no significant bias in angle distribution for either cue (Rayleigh test for uniformity, Shh: n = 2,028; Netrin: n = 2,805). Scale bars (B-D): 10 μm. (G,H): 25 μm. Error bars represent SEM. cb, cell body; gc, growth cone. See also S3 Fig.
Mentions: We established gradients of Shh or Netrin-1 after commissural neurons had been cultured for 24 h, when the majority of neurons had already initiated an axon. We calculated the turned angle of an axon as the difference between the base and tip angles (Fig. 3A) and scored the angle as positive if the axon turned towards the gradient and negative if it turned away. By varying the maximal concentration of ligand in a particular chamber, we could test a wide range of concentrations. In a control gradient (Phosphate buffered saline/BSA), we observed a wide range of turned angles towards and away from the gradient, resulting in a net turned angle of 0° (Fig. 3B,E). Neurons exposed to a gradient of Shh (Fig. 3C,E) or Netrin-1 (Fig. 3D,F), however, turned towards the higher concentration of chemoattractant. For either cue, we observed axon turning in response to a wide range of concentrations at the growth cone (Fig. 3E,F). The distribution of turned angles of individual axons confirmed that wide concentrations of Shh and Netrin-1 induced biases towards attraction (Fig. 3G,H). To eliminate the possibility that Shh or Netrin-1 influences the orientation at which the axon exits the cell body (axon base angle), thus confounding our measurement of the angle turned, we performed experiments where gradients were established 4–6 h after the neurons were plated, before the majority of neurons had initiated an axon. We found that Shh and Netrin-1 gradients induced no significant bias in the distribution of axon base angles facing up-gradient (higher concentration) compared to those facing down-gradient (lower concentration) (Fig. 3I,J). Therefore, le Massif generates gradients that can induce axon turning without any effect on axonal initiation.

Bottom Line: We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow.We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1.Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada; Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada; Program in Neuroengineering, McGill University, Montreal, Quebec, Canada.

ABSTRACT
During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.

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