Limits...
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.

Show MeSH
le Massif microfluidic gradient generator can produce shallow linear concentration gradients that are stable through space and time.(A) Drawing of the le Massif microfluidic device. Dimensions of the glass slide and the cell culture chamber are indicated with dashed lines. The blue line represents the upstream limit of the gradient chamber; the red line represents the downstream limit. The device generates linear concentration gradients using a pre-mixing microfluidic paradigm, in which a known concentration of cue is added to a reservoir at inlet 1 and culture media without guidance cue is added to inlet 2. (B) Schematic cross-section of le Massif microfluidic gradient generator. The fluid-filled inlet reservoirs drive a flow from left to right, with the fluid accumulating in the hole at the outlet. (C–E) The gradient visualized using tetramethylrhodamine-conjugated 40 kDa dextran. When the streams from inlets 1 and 2 converge, the concentrations are mixed and divided at 18 discrete steps throughout the premixers (C), generating 20 discrete concentrations that then enter into the gradient chamber (D). The overall gradient shape smoothens rapidly by diffusion to become continuous while maintaining its overall profile until the downstream region (red line in E), where the media flows to the outlet. (F,G) Theoretical calculation of the concentration (F) and fractional change in concentration, δ, (G) of guidance cue in the gradient chamber. The fractional change range (0.3 ≤ δ < 2.2%) encompasses the shallow gradients observed in vivo. (H,I) Measured fluorescence intensity (H) of the upstream (blue line in D) and downstream (red line in E) limits of the gradient chamber, as well as the fractional change in concentration (I) calculated from the upstream concentration profile. Mean ± standard error of the mean (SEM) of 14 independent gradient devices are shown. The measured values match the theoretical predictions (green line in I), with a mean-square-error of 0.091 compared with the theoretical fractional change. (J,K) Gradient profile over time for the upstream (J) and downstream (K) limits. The red boxes in (K) represent 450 μm, which are excluded due to the gradient flattening that occurs at the boundaries of the device resulting from the no-slip condition. (L) Stitched inverted fluorescence image of the downstream area shown in (E), seeded with commissural neurons that were fluorescently stained with rhodamine-conjugated phalloidin. (M) Representative higher-magnification inverted fluorescence image of rhodamine-phalloidin-stained commissural neurons in the gradient chamber. After 45 h in culture, most neurons have extended an axon. Scale bar (C–E,L): 1 mm. (M): 25 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4380419&req=5

pbio.1002119.g002: le Massif microfluidic gradient generator can produce shallow linear concentration gradients that are stable through space and time.(A) Drawing of the le Massif microfluidic device. Dimensions of the glass slide and the cell culture chamber are indicated with dashed lines. The blue line represents the upstream limit of the gradient chamber; the red line represents the downstream limit. The device generates linear concentration gradients using a pre-mixing microfluidic paradigm, in which a known concentration of cue is added to a reservoir at inlet 1 and culture media without guidance cue is added to inlet 2. (B) Schematic cross-section of le Massif microfluidic gradient generator. The fluid-filled inlet reservoirs drive a flow from left to right, with the fluid accumulating in the hole at the outlet. (C–E) The gradient visualized using tetramethylrhodamine-conjugated 40 kDa dextran. When the streams from inlets 1 and 2 converge, the concentrations are mixed and divided at 18 discrete steps throughout the premixers (C), generating 20 discrete concentrations that then enter into the gradient chamber (D). The overall gradient shape smoothens rapidly by diffusion to become continuous while maintaining its overall profile until the downstream region (red line in E), where the media flows to the outlet. (F,G) Theoretical calculation of the concentration (F) and fractional change in concentration, δ, (G) of guidance cue in the gradient chamber. The fractional change range (0.3 ≤ δ < 2.2%) encompasses the shallow gradients observed in vivo. (H,I) Measured fluorescence intensity (H) of the upstream (blue line in D) and downstream (red line in E) limits of the gradient chamber, as well as the fractional change in concentration (I) calculated from the upstream concentration profile. Mean ± standard error of the mean (SEM) of 14 independent gradient devices are shown. The measured values match the theoretical predictions (green line in I), with a mean-square-error of 0.091 compared with the theoretical fractional change. (J,K) Gradient profile over time for the upstream (J) and downstream (K) limits. The red boxes in (K) represent 450 μm, which are excluded due to the gradient flattening that occurs at the boundaries of the device resulting from the no-slip condition. (L) Stitched inverted fluorescence image of the downstream area shown in (E), seeded with commissural neurons that were fluorescently stained with rhodamine-conjugated phalloidin. (M) Representative higher-magnification inverted fluorescence image of rhodamine-phalloidin-stained commissural neurons in the gradient chamber. After 45 h in culture, most neurons have extended an axon. Scale bar (C–E,L): 1 mm. (M): 25 μm.

Mentions: We thus developed a guidance assay capable of simulating, over 1–2 d, the shallow Shh gradients that we observed in the spinal cord in vivo. Microfluidic mixing networks allow gradients to be controlled in space and time, allowing for long-term gradients to be established, in contrast to passive source-sink diffusion gradients (e.g., pipette assay and Dunn chamber). We used a linear gradient generator because it allowed us to test a range of fractional change (δ) values. We modified a pre-mixer microfluidic gradient generator [23] by increasing both the length and width of the gradient region, thus maximizing the surface area on which neurons could be exposed to the gradient and thus the sample size. By increasing the width of the gradient, we also decreased the range of gradient steepness to physiologically relevant levels, as determined in vivo (Fig. 1B). Our wider gradient chamber required an increase in the number of sequential mixing channels (Fig. 2A), which offered the added benefit of increasing the overall resistance, thus decreasing the flow velocity and resulting shear stress, which can be harmful to axons [24]. With these device improvements, we were thus able to generate stable, long-term gradients.


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)

le Massif microfluidic gradient generator can produce shallow linear concentration gradients that are stable through space and time.(A) Drawing of the le Massif microfluidic device. Dimensions of the glass slide and the cell culture chamber are indicated with dashed lines. The blue line represents the upstream limit of the gradient chamber; the red line represents the downstream limit. The device generates linear concentration gradients using a pre-mixing microfluidic paradigm, in which a known concentration of cue is added to a reservoir at inlet 1 and culture media without guidance cue is added to inlet 2. (B) Schematic cross-section of le Massif microfluidic gradient generator. The fluid-filled inlet reservoirs drive a flow from left to right, with the fluid accumulating in the hole at the outlet. (C–E) The gradient visualized using tetramethylrhodamine-conjugated 40 kDa dextran. When the streams from inlets 1 and 2 converge, the concentrations are mixed and divided at 18 discrete steps throughout the premixers (C), generating 20 discrete concentrations that then enter into the gradient chamber (D). The overall gradient shape smoothens rapidly by diffusion to become continuous while maintaining its overall profile until the downstream region (red line in E), where the media flows to the outlet. (F,G) Theoretical calculation of the concentration (F) and fractional change in concentration, δ, (G) of guidance cue in the gradient chamber. The fractional change range (0.3 ≤ δ < 2.2%) encompasses the shallow gradients observed in vivo. (H,I) Measured fluorescence intensity (H) of the upstream (blue line in D) and downstream (red line in E) limits of the gradient chamber, as well as the fractional change in concentration (I) calculated from the upstream concentration profile. Mean ± standard error of the mean (SEM) of 14 independent gradient devices are shown. The measured values match the theoretical predictions (green line in I), with a mean-square-error of 0.091 compared with the theoretical fractional change. (J,K) Gradient profile over time for the upstream (J) and downstream (K) limits. The red boxes in (K) represent 450 μm, which are excluded due to the gradient flattening that occurs at the boundaries of the device resulting from the no-slip condition. (L) Stitched inverted fluorescence image of the downstream area shown in (E), seeded with commissural neurons that were fluorescently stained with rhodamine-conjugated phalloidin. (M) Representative higher-magnification inverted fluorescence image of rhodamine-phalloidin-stained commissural neurons in the gradient chamber. After 45 h in culture, most neurons have extended an axon. Scale bar (C–E,L): 1 mm. (M): 25 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380419&req=5

pbio.1002119.g002: le Massif microfluidic gradient generator can produce shallow linear concentration gradients that are stable through space and time.(A) Drawing of the le Massif microfluidic device. Dimensions of the glass slide and the cell culture chamber are indicated with dashed lines. The blue line represents the upstream limit of the gradient chamber; the red line represents the downstream limit. The device generates linear concentration gradients using a pre-mixing microfluidic paradigm, in which a known concentration of cue is added to a reservoir at inlet 1 and culture media without guidance cue is added to inlet 2. (B) Schematic cross-section of le Massif microfluidic gradient generator. The fluid-filled inlet reservoirs drive a flow from left to right, with the fluid accumulating in the hole at the outlet. (C–E) The gradient visualized using tetramethylrhodamine-conjugated 40 kDa dextran. When the streams from inlets 1 and 2 converge, the concentrations are mixed and divided at 18 discrete steps throughout the premixers (C), generating 20 discrete concentrations that then enter into the gradient chamber (D). The overall gradient shape smoothens rapidly by diffusion to become continuous while maintaining its overall profile until the downstream region (red line in E), where the media flows to the outlet. (F,G) Theoretical calculation of the concentration (F) and fractional change in concentration, δ, (G) of guidance cue in the gradient chamber. The fractional change range (0.3 ≤ δ < 2.2%) encompasses the shallow gradients observed in vivo. (H,I) Measured fluorescence intensity (H) of the upstream (blue line in D) and downstream (red line in E) limits of the gradient chamber, as well as the fractional change in concentration (I) calculated from the upstream concentration profile. Mean ± standard error of the mean (SEM) of 14 independent gradient devices are shown. The measured values match the theoretical predictions (green line in I), with a mean-square-error of 0.091 compared with the theoretical fractional change. (J,K) Gradient profile over time for the upstream (J) and downstream (K) limits. The red boxes in (K) represent 450 μm, which are excluded due to the gradient flattening that occurs at the boundaries of the device resulting from the no-slip condition. (L) Stitched inverted fluorescence image of the downstream area shown in (E), seeded with commissural neurons that were fluorescently stained with rhodamine-conjugated phalloidin. (M) Representative higher-magnification inverted fluorescence image of rhodamine-phalloidin-stained commissural neurons in the gradient chamber. After 45 h in culture, most neurons have extended an axon. Scale bar (C–E,L): 1 mm. (M): 25 μm.
Mentions: We thus developed a guidance assay capable of simulating, over 1–2 d, the shallow Shh gradients that we observed in the spinal cord in vivo. Microfluidic mixing networks allow gradients to be controlled in space and time, allowing for long-term gradients to be established, in contrast to passive source-sink diffusion gradients (e.g., pipette assay and Dunn chamber). We used a linear gradient generator because it allowed us to test a range of fractional change (δ) values. We modified a pre-mixer microfluidic gradient generator [23] by increasing both the length and width of the gradient region, thus maximizing the surface area on which neurons could be exposed to the gradient and thus the sample size. By increasing the width of the gradient, we also decreased the range of gradient steepness to physiologically relevant levels, as determined in vivo (Fig. 1B). Our wider gradient chamber required an increase in the number of sequential mixing channels (Fig. 2A), which offered the added benefit of increasing the overall resistance, thus decreasing the flow velocity and resulting shear stress, which can be harmful to axons [24]. With these device improvements, we were thus able to generate stable, long-term gradients.

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.

Show MeSH