Koser DE et al. (2016), Mechanosensing is critical for axon growth in t...

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Nat Neurosci 2016 Dec 01;1912:1592-1598. doi: 10.1038/nn.4394.

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Mechanosensing is critical for axon growth in the developing brain.

Koser DE , Thompson AJ , Foster SK , Dwivedy A , Pillai EK , Sheridan GK , Svoboda H , Viana M , Costa LD , Guck J , Holt CE , Franze K .

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During nervous system development, neurons extend axons along well-defined pathways. The current understanding of axon pathfinding is based mainly on chemical signaling. However, growing neurons interact not only chemically but also mechanically with their environment. Here we identify mechanical signals as important regulators of axon pathfinding. In vitro, substrate stiffness determined growth patterns of Xenopus retinal ganglion cell axons. In vivo atomic force microscopy revealed a noticeable pattern of stiffness gradients in the embryonic brain. Retinal ganglion cell axons grew toward softer tissue, which was reproduced in vitro in the absence of chemical gradients. To test the importance of mechanical signals for axon growth in vivo, we altered brain stiffness, blocked mechanotransduction pharmacologically and knocked down the mechanosensitive ion channel piezo1. All treatments resulted in aberrant axonal growth and pathfinding errors, suggesting that local tissue stiffness, read out by mechanosensitive ion channels, is critically involved in instructing neuronal growth in vivo.

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Species referenced: Xenopus laevis
Genes referenced: fn1 piezo1

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	Figure 1. Mechanosensitivity of RGC axons in vitro. (a, b) Cultures of Xenopus eye primordia (asterisks) on (a) âsoftâ (0.1 kPa) and (b) âstiffâ (1 kPa) substrates. Arrows indicate axons. (c) Eye primordium grown on a stiff substrate and treated with GsMTx4, a blocker of mechanosensitive ion channels. Scale bar: 200 Âµm. (d) Sholl analysis of axon lengths after 24 hours (normalized counts as mean Â± S.E.M.). (e) Median distances shown in (d). Axons were significantly longer on stiffer substrates than either on soft ones (One-Way-ANOVA followed by Bonferroni post-hoc test; P = 2.79 Ã 10-7, t = 6.354,) or after GsMTx4 treatment (P = 5.01 Ã 10-8, t = -6.855). Neurons grown on stiff substrates and treated with GsMTx4 resembled neurons grown on soft substrates (P = 1.00, t = 0.082). n = number of eye primordia from three biological replicates. (f) Immunocytochemistry showing f-actin (green), beta-tubulin (red) and the mechanosensitive ion channel Piezo1 (white). Scale bar: 10 Âµm. (g) The extension velocity of axons was higher on stiff substrates (Mann-Whitney-Test; P = 9.32 Ã 10-6, Z = 4.432). (h) On soft substrates, growth cones explored their environment more and migrated significantly faster than on stiff ones (two-tailed t-test; P = 0.00867, t = 2.669). (i) On stiff substrates, axon growth was more directed (i.e., straight) than on soft substrates (Mann-Whitney-Test; P = 1.10 Ã 10-6, Z = 4.873). n = number of axons from three biological replicates. (j) Processed fluorescence images of beta-tubulin-labelled RGC axons; colour represents local angular orientation of axonal segments. On soft substrates, axons grew less directionally persistent (from bottom to top; cf. Supplementary Fig. 2e-g). Scale bar: 15 Âµm. All experiments were repeated three times, and representative images are shown. Boxes show the 25th, 50th (the median), and 75th percentiles, whiskers the spread of the data.
	Figure 2. In vivo brain mechanics. (a) Schematic of the experimental setup. (b) Xenopus brain. The dashed red line indicates the stiffness map area. (c, d) Images of Xenopus embryos with overlaid AFM-based stiffness maps of exposed in vivo brain tissue. Colour encodes the apparent elastic modulus K assessed at an indentation force of 7 nN. Blue shape in (d) shows the OT location (based on fluorescence images, Supplementary Fig. 3). Scale bar: 200 Âµm. At both stage 33/34 (c) and stages 39-40 (d), brain tissue was mechanically heterogeneous and displayed clearly visible stiffness gradients. Green dashed lines indicate tectum boundaries. The grey dashed square in (d) indicates a region as shown in (e). (e) Immunohistochemistry demonstrated a significantly higher density of cell nuclei (blue) rostral to the OT (yellow) than caudal to it. Scale bar: 20 Âµm. (f) The tectum was softer than the tel-/diencephalon at stage 33/34 (Mann-Whitney-Test; P = 2.26 Ã 10-9, Z = 5.978) and (g) than the OT at stages 39-40 (P = 0.0033, Z = 2.933). (h) At stages 39-40, tissue rostral of the OT was significantly stiffer than caudal of it (P = 2.97 Ã 10-5, Z = 4.163). (i) Quantification of cell density on both sides of the OT; cell density was significantly higher rostral to the OT (paired two-tailed t-test; P = 3.96 Ã 10-6, t = 9.879). n = number of measurements, N = animal numbers. All representative images and stiffness maps shown are from three biological replicates. Boxes show the 25th, 50th (the median), and 75th percentiles, whiskers the spread of the data.
	Figure 3. Neurons grow towards soft tissue. (a) Schematic showing how local gradients in brain tissue stiffness perpendicular to the RGC axon growth direction M and the local OT curvature C were determined. (b) Relationship between M and C. (c) Same data as in (b), pooled. Axons in vivo preferentially turned towards the softer side of the tissue (one-sample Wilcoxon Signed Rank Test; P = 1.44 Ã 10-4,Z = 3.801). n = number of measurement points from 7 animals. (d, e) Time-lapse imaging of individual axon bundles growing on a stiffness gradient matching that found in vivo (Mmax â¼ 2 Pa/Âµm; cf. Supplementary Fig. 1) revealed that also in vitro, in the absence of chemical gradients, RGC axons preferentially turned towards the softer side of the substrate (one-sample Wilcoxon Signed Rank Test; P = 0.0549; Z = -1.913). Scale bar: 20 Âµm. Representative images from 11 biological replicates are shown. (f) Eye primordium cultured on a similar stiffness gradient (indicated by colour). Axons growing more clockwise in the left half and more counter-clockwise in the right half of the image turned towards the soft side of the substrate. Scale bar: 200 Âµm. The experiment was repeated three times, and a representative image is shown. (g) Quantification of individual axon segment orientations similarly revealed preferential turning towards the soft side of the substrate (one-sample Wilcoxon Signed Rank Test; P = 0.0264, Z = 2.197). Boxes show the 25th, 50th (the median), and 75th percentiles, whiskers the spread of the data.
	Figure 4. Changing brain stiffness leads to axonal pathfinding errors. (a) Xenopus brain treated with 15 mg/ml CS, overlaid with an AFM-based stiffness map. Blue curve indicates OT location (based on fluorescence images, Supplementary Fig. 3), green dashed curve tectum boundary, grey dashed line the region in front of the OT. Scale bar: 200 Âµm. (b) Tissue was significantly stiffer in controls (blue) compared to CS-treated brains (green) (Mann-Whitney-Test; P = 6.62 Ã 10-10, Z = 6.175), particularly in front of the OT (c) (P = 8.57 Ã 10-11, Z = 6.490). n = number of measurements, N = animal numbers. (d) Image of CS-treated, softened brain. The dashed black curve indicates the outline of the OT. RGC axons dispersed widely from their normal trajectory. Scale bar: 100 Âµm. (e) Example of a fit of an ellipse around the outline of an OT; the ratio of long and short axis determines the elongation. (f) CS treatment significantly decreased the elongation of the OT (two-tailed t-test; P = 3.12 Ã 10-6, t = 5.462). n = number of animals. (g) Schematic illustration of local mechanical brain manipulation. Soft tissue caudal to the presumptive caudal turn of the OT was locally indented with an AFM cantilever for â¼6 hours. (h) In control brains, the OT grew normally. (i) When a force was locally exerted on the tissue (position of the probe is indicated), axons grew away from the cantilever probe, thus deviating from their normal pathway. Inset: magnification of the distal part of the OT. Scale bar: 100 Âµm. All experiments were repeated three times, and representative images are shown. Boxes show the 25th, 50th (the median), and 75th percentiles, whiskers the spread of the data.
	Figure 5. In vivo manipulation of mechanosensitive ion channels disrupts axon pathfinding. (a-d) The application of 25 ÂµM GsMTx4 disrupted axon pathfinding in vivo. Axons were shorter and spread more, resembling axons cultured on soft substrates in vitro. (c-d) Enlarged boxes shown in (a) and (b). (e-h) Similarly, MO knockdown of Piezo1 led to aberrant axon growth in vivo. Scale bars: 100 Âµm. Experiments were repeated three (a-d) or four times (e-h), and representative images are shown. (i) Quantification of OT morphology. Interfering with mechanotransduction led to a significantly decreased elongation of the OT. (two-tailed t-test; PGsMTx4 = 0.00243, t = 3.231; PPiezo1-MO = 0.00476, t = 2.932). Phenotypes of controls were similar (P = 0.782, t = 0.278), as well as those of GsMTx4-treated and Piezo1 knockdown animals (P = 0.0976, t = 1.692). n = number of animals. Boxes show the 25th, 50th (the median), and 75th percentiles, whiskers the spread of the data.
	Figure 6. Schematics of the mechanical control of axon growth. (a) Schematic summary of this study. Shown is the outline of a Xenopus brain and the OT. Mechanical signals contribute to neuronal growth in the developing CNS. RGC axons grow faster, straighter, and more parallel on stiffer than on softer substrates (Fig. 1). Accordingly, the part of the brain these axons have to pass is stiffer than the tectum (Fig. 2), where axon growth slows down and eventually stops. The softness of the tectum then facilitates unbundling and branching. On their way, axons encounter an area with a stiffness gradient, which contributes to the turning of the OT towards the softer side of the brain (Fig. 3). (b) Schematic illustration of a mechanism by which axon bundles encountering a perpendicular stiffness gradient might turn towards the softer side of the substrate. The velocity of axons is larger on stiffer substrates (upper panel). As axons in the OT fasciculate, they are mechanically coupled, so the faster axons growing on the stiffer side may be âpulledâ towards the slower axons on the softer side, leading to a reorientation of the axon bundle and overall growth towards the softer side.
	Supplementary Figure 1. In vitro stiffness gradient culture substrates. (a) Description of chamber assembly. Premixes of stiff and soft gels were prepared (with fluorescently tagged stiff polymer), polymerization was initiated, polymerizing stiff gel filled into the chamber (to fill half of it), which was then overlaid with polymerizing soft gel. At the interface between the two solutions, polymerizing gels mixed due to diffusion, resulting in a gradient in stiffness and fluorescence signal in the polymerized gel. (b) AFM stiffness map of a gel stiffness gradient44. (c) Laminin coating of PAA gels. Immunofluorescence showed equal coating densities on soft and stiff gels, indicating homogeneous coating of the substrate surface (Mann-Whitney-Test; P = 0.430, Z = 0.789). n = number of randomly selected regions of interest from three independent experiments. (d) Quantification of stiffness gradients. Stiffness gradients were sigmoidal, with a large (~3 mm) linear region (quality of a linear fit to the linear region: R2 = 0.99 Â± 0.01 (mean Â± S.E.M.), n = 8). The average slope of the stiffness gradients was Mmax = (1.8 Â± 0.3) Pa/Âµm (nÂ =Â 8). (e) There was a strong correlation between local substrate stiffness and fluorescence intensity (Pearsonâs correlation coefficient r > 0.98 for all measured substrates (n = 4)), enabling the use of fluorescence signal as readout for K.
	Supplementary Figure 2. Quantification of axon growth on compliant culture substrates. (a-d) Axon growth on compliant substrates coated with fibronectin. (a, b) Cultures of Xenopus eye primordia (asterisks) on (a) âsoftâ (0.1 kPa) and (b) âstiffâ (1 kPa) substrates. Shown are representative images from three independent experiments. (c) Sholl analysis of axon lengths after 24 hours (normalized counts as mean Â± S.E.M.). (d) Average of the median distances shown in (c). Axons were significantly longer on stiffer substrates than on soft ones (two-tailed t-test; P = 1.82 Ã 10-6, t = 6.118). Although fibronectin engages different integrins than laminin, RGC axons assumed similar phenotypes on soft and stiff substrates in these experiments, indicating that neurons were mechanosensitive irrespective of the type of integrins involved in cell adhesion. n = number of eye primordia from three biological replicates. (e-g) Directionality of axon growth on compliant substrates. RGC axon growth directions are more random on soft (e) than on stiff (f) substrates. Rose diagrams show the distribution of turning angles of growth cones. 0Â° corresponds to a straight forward movement, 90Â° to a left-turn, and 180Â° corresponds to retractions. Each ring in the rose plot represents the labelled percentage of the total number of angles. The proportion of axons growing straight forward is significantly larger on stiffer substrates than on softer ones (for axons growing between 355Â° and 5Â°: P < 10-3, Mann-Whitney-test, n = 60 each for soft and stiff). (g) Quantification of Fig. 1j. A significantly larger percentage of axons was deviating by more than 20Â° from a straight path on soft compared to stiff substrates (two-tailed t-test; P = 0.026, t = 2.274), showing that axons grew less ordered on softer substrates. nÂ =Â number of randomly selected regions of interest from three independent experiments.
	Supplementary Figure 3. Elasticity maps for a fixed indentation depth. a, b, d) Same samples as shown in Figs. 2c, d, and 4a, respectively; here K was determined not at a defined force but at a defined indentation depth ï¤ = 3Âµm. Stiffness gradients in the tissue are very similar to those shown in Figs. 2 and 4. Blue lines indicate outlines of the OT. Insets in (b) and (d): epifluorescence images of RGC axons in the OT of the respective brains. (c) Stiffness map of a brain with ablated RGC axons. Similar stiffness gradients are found as in control brains, indicating that gradients likely arise from the distribution of cell somata in the tissue (cf. Fig. 2e). Scale bar: 200 Âµm. Shown are representative images and stiffness maps from three independent experiments.

References [+] :

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