Mechanosensing is critical for axon growth in the developing brain

Abstract

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.

Figure 1. Mechanosensitivity of RGC axons in vitro.

(a, b) Cultures of Xenopus eye primordia (asterisks) on (a) ‘soft’ (0.1 kPa) and (b) ‘stiff’ (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 µm. (d) Sholl analysis of axon lengths after 24 hours (normalized counts as mean ± 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 × 10^-7, t = 6.354,) or after GsMTx4 treatment (P = 5.01 × 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 µm. (g) The extension velocity of axons was higher on stiff substrates (Mann-Whitney-Test; P = 9.32 × 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 × 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 µm. All experiments were repeated three times, and representative images are shown. Boxes show the 25^th, 50^th (the median), and 75^th 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 µm. 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 µm. (f) The tectum was softer than the tel-/diencephalon at stage 33/34 (Mann-Whitney-Test; P = 2.26 × 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 × 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 × 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 25^th, 50^th (the median), and 75^th 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 × 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 (M_max ∼ 2 Pa/µm; 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 µm. 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 µm. 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 25^th, 50^th (the median), and 75^th 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 µm. (b) Tissue was significantly stiffer in controls (blue) compared to CS-treated brains (green) (Mann-Whitney-Test; P = 6.62 × 10^-10, Z = 6.175), particularly in front of the OT (c) (P = 8.57 × 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 µm. (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 × 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 ∼6 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 µm. All experiments were repeated three times, and representative images are shown. Boxes show the 25^th, 50^th (the median), and 75^th 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 µM 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 µm. 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; P_GsMTx4 = 0.00243, t = 3.231; P_Piezo1-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 25^th, 50^th (the median), and 75^th 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 ‘pulled’ towards the slower axons on the softer side, leading to a reorientation of the axon bundle and overall growth towards the softer side.