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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We thank D. Bray, A. Reichenbach, B. Simons and S. Wolff for discussions about neuronal mechanics, K. Chalut and W. Harris for feedback on the manuscript, A. Christ for an AFM data analysis code, P. Moshayedi for help with establishing PAA gels, F. Sachs (University at Buffalo, USA) for providing GsMTx4, H. Wong for extensive help in the lab and A. Winkel and R. Field (JPK) for technical help. This work was supported by the German National Academic Foundation (scholarship to D.E.K.), Wellcome Trust and Cambridge Trusts (scholarships to A.J.T.), Winston Churchill Foundation of the United States (scholarship to S.K.F.), Herchel Smith Foundation (Research Studentship to S.K.F.), CNPq 307333/2013-2 (L.d.F.C.), NAP-PRP-USP and FAPESP 11/50761-2 (L.d.F.C.), UK EPSRC BT grant (J.G.), Wellcome Trust WT085314 and the European Research Council 322817 grants (C.E.H.); an Alexander von Humboldt Foundation Feodor Lynen Fellowship (K.F.), UK BBSRC grant BB/M021394/1 (K.F.), the Human Frontier Science Program Young Investigator Grant RGY0074/2013 (K.F.), the UK Medical Research Council Career Development Award G1100312/1 (K.F.) and the Eunice Kennedy Shriver National Institute Of Child Health & Human Development of the National Institutes of Health under Award Number R21HD080585 (K.F.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The authors declare no competing financial interests.
Integrated supplementary information
(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 gradient. (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 ± 0.01 (mean ± S.E.M.), n = 8). The average slope of the stiffness gradients was Mmax = (1.8 ± 0.3) Pa/µm (n = 8). (e) There was a strong correlation between local substrate stiffness and fluorescence intensity (Pearson’s correlation coefficient r > 0.98 for all measured substrates (n = 4)), enabling the use of fluorescence signal as readout for K.
(a-d) Axon growth on compliant substrates coated with fibronectin. (a, b) Cultures of Xenopus eye primordia on (a) ‘soft’ (0.1 kPa) and (b) ‘stiff’ (1 kPa) substrates. Shown are representative images from three independent experiments. (c) Sholl analysis of axon lengths after 24 hours (normalized counts as mean ± S.E.M.). (d) Box plots 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 × 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 independent experiments. (e-g) Directionality of axon growth on compliant substrates coated with laminin. 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. 0° corresponds to a straight forward movement, 90° to a left-turn, and 180° 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 355° and 5°: P = 3.88 x 10-4, z = 3.548, 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 20° 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. n = number of randomly selected regions of interest from three independent experiments.
(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 δ = 3 µm. 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 µm. Experiments were repeated three times and representative images are shown.
(a) Relationship between local gradients in tissue stiffness perpendicular to the RGC axon growth direction, M, and local OT curvature, C, in Xenopus brains treated with 15 mg/ml CS. (b) Same data as in (a), pooled (green). Blue box plot (controls) replotted from Fig. 3c for comparison. Also in CS-treated brains, axons in vivo preferentially turned towards the softer side of the tissue (one-sample Wilcoxon Signed Rank Test; P = 5.63 × 10-4, Z = 3.449). There was no statistical difference between control and CS-treated animals (Mann-Whitney-Test; P = 0.354, Z = 0.927). n = number of measurement points from nine CS-treated animals. (c, d) Relationship between the absolute stiffness K of the gradient RGC axons encountered in vivo and curvature C of the OT. We did not find a correlation between K and curvature in either control (c) or CS-treated (d) brains; only the gradient strength M is important (cf. Fig. 3).
(a) Image of an AFM experiment. A sustained stress was applied with a cantilever, likely leading to a stiffening of the tissue underneath the spherical probe (outline indicated by black circle). Axons (surrounded by dashed curve, arrow) had started growing along the brain surface but had not reached the mid-diencephalon yet. Scale bar: 200 µm. (b,c,f,g) Images showing outlines of Xenopus brains and the OT (outlined by dashed curve) at the end of four independent experiments. The position of the AFM probe is shown by a filled circle. Axons avoided the tissue underneath the bead and in some cases (b, c) grew around the stiffened tissue. (d) and (e) show magnified regions indicated by dashed rectangles in (b) and (c), respectively. Dashed circles indicate AFM probe positions. Scale bars: 100 µm. (h) Compression led to significant tissue stiffening (n = 13 from four different animals, Kruskal-Wallis ANOVA; P = 0.00683, χ2 = 9.97396).
(a-c) MO knockdown of Piezo1 led to aberrant axon growth in vivo, which was maintained at stage 42 (~1 day after the experiments shown in Fig. 5). Scale bar: 100 µm. Representative images from three independent experiments. (c) Quantification of OT morphology. At stage 42, elongation was still significantly decreased (two-tailed t-test; P = 5.21 × 10-4; t = 3.902), showing that the knockdown did not merely slow down axon growth but rather led to a ‘soft phenotype’ with shorter RGC axons that deviated from their normal path. n = number of animals from three experimental days. (d, e) Western blot analysis confirmed that Piezo1 morpholinos led to a significant decrease in the expression of the protein by 42 % ± 9 % if compared to the scrambled control (n = 4 Western blots from 2 lysates; One-sample t-test; P = 0.00689, t = 6.634). (d) Cropped lanes of Piezo1 and a-tubulin. (e) Full-length blot with molecular weight standard.
Supplementary Figures 1–6 (PDF 967 kb)
Time-lapse movie of Xenopus retinal ganglion cell axons cultured on a soft substrate of G' = 100 Pa.
1 s corresponds to 3.5 min of real time. Scale bar = 25 μm. (AVI 3960 kb)
Time-lapse movie of Xenopus retinal ganglion cell axons cultured on a soft substrate of G' = 1,000 Pa.
1 s corresponds to 3.5 min of real time. Scale bar = 25 μm. (AVI 1811 kb)
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Koser, D., Thompson, A., Foster, S. et al. Mechanosensing is critical for axon growth in the developing brain. Nat Neurosci 19, 1592–1598 (2016). https://doi.org/10.1038/nn.4394
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