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Nature Neuroscience | Article

YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells

Journal name:
Nature Neuroscience
Volume:
19,
Pages:
879–887
Year published:
DOI:
doi:10.1038/nn.4316
Received
Accepted
Published online

Abstract

Myelination is essential for nervous system function. Schwann cells interact with neurons and the basal lamina to myelinate axons using known receptors, signals and transcription factors. In contrast, the transcriptional control of axonal sorting and the role of mechanotransduction in myelination are largely unknown. Yap and Taz are effectors of the Hippo pathway that integrate chemical and mechanical signals in cells. We describe a previously unknown role for the Hippo pathway in myelination. Using conditional mutagenesis in mice, we show that Taz is required in Schwann cells for radial sorting and myelination and that Yap is redundant with Taz. Yap and Taz are activated in Schwann cells by mechanical stimuli and regulate Schwann cell proliferation and transcription of basal lamina receptor genes, both necessary for radial sorting of axons and subsequent myelination. These data link transcriptional effectors of the Hippo pathway and of mechanotransduction to myelin formation in Schwann cells.

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  1. Yap and Taz expression and activation during Schwann cell development.
    Figure 1: Yap and Taz expression and activation during Schwann cell development.

    (a) Yap and Taz staining (green) of Schwann cells plated on neurons for times indicated in myelinating (ascorbic acid, AA) or nonmyelinating (no AA) conditions. NF, neurofilament staining; DAPI, nuclear staining; phalloidin, F actin staining; Mbp, myelin basic protein. Arrowheads indicate detection of Yap and Taz in the nucleus of Schwann cells at low density; arrows indicate nuclei devoid of Yap and Taz in myelinating Schwann cells. Insets, one cell stained for Yap and Taz in each condition and enlarged. The experiment was repeated twice on at least 3 coverslips per repeat. Scale bar, 20 μm. (b) Western blot analysis of Yap and Taz expression during development and myelination in sciatic nerve lysates. Cnx, calnexin. The experiment was performed once (full-length blots are presented in Supplementary Fig. 4). (c) Teased fibers from P20 and P40 sciatic nerve stained for Yap (green), DAPI (blue) and NF (red) show that Yap is nuclear (arrowheads indicate nuclei) after myelination. The experiment was repeated twice. Scale bar, 20 μm.

  2. Laminins and mechanical stimulation regulate Yap and Taz in primary Schwann cells.
    Figure 2: Laminins and mechanical stimulation regulate Yap and Taz in primary Schwann cells.

    (a–c) Confocal immunofluorescence images of Yap and Taz (green), phalloidin (red) and DAPI (blue) in Schwann cells plated at different densities and treated or not with blebbistatin (BBS, 25 μM) (a) or plated sparsely on polyacrylamide (0.5 kPa, 40 kPa), PDMS (4 MPa) or glass (4 GPa) (b) or on polyacrylamide plus laminin 211 (c). (d) Confocal immunofluorescence images of Yap and Taz (green), phalloidin (red) and DAPI (blue) in Schwann cells plated on silicone substrate with or without laminin 211 and stretched for 30 min. Arrowheads indicate cytosolic Yap and Taz; asterisks indicate nuclear Yap and Taz. (e) Quantification of nuclear and cytoplasmic Yap and Taz scored in >500 cells (777 unstretched, 531 stretched). ****P < 0.0001, Fisher's exact test. Lm211, laminin 211. Scale bars, 20 μm. Experiments were repeated two (a) or three (b–e) times on a minimum of three samples.

  3. Ablation of Taz in Schwann cells impairs radial sorting of axons.
    Figure 3: Ablation of Taz in Schwann cells impairs radial sorting of axons.

    (a) Western blot analysis of Taz and Yap expression in sciatic nerves of Taz and Yap cKO mice. The experiment was repeated twice (full-length blots are presented in Supplementary Fig. 4). Cnx, calnexin. (b–d) Toluidine-blue-stained semithin cross-sections of sciatic nerves from control (b) Yap cKO (c) and Taz cKO (d) mice at P20. Arrows indicate bundles of unsorted axons. Scale bar, 20 μm. Three mice per genotype were analyzed. (e) Myelin thickness as measured by g-ratio in littermate control, Taz cKO, Yap cKO and Yap cKO–Taz cHet mice. Each data point indicates the average value from one nerve from a different animal. Error bars indicate mean and s.e.m. n = 3 mice per group. One-way ANOVA. (f–m) Toluidine-blue-stained semithin cross-sections (f–i) and electron micrographs (j–m) of sciatic nerves from control (f,j), Taz cKO–Yap cHet (g,k); Taz cHet–Yap cKO (h,l); and double-cKO (i,m) mice at P20. Scale bars, 10 μm (b–d), 20 μm (f–i) or 2 μm (j–m). Myelinating Schwann cell (asterisks), promyelinating Schwann cells (arrows) and immature Schwann cells (arrowheads) are indicated in g, h, k and l. (n,o) Quantification of myelinated (n) and amyelinated (o) fibers in control and mutant mice at P20. Data are presented as mean ± s.e.m. Each data point indicates the average value from one nerve from a different mouse; n = 2 (double cKO) or 3 mice per genotype (all others). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001, two-tailed unpaired Student's t-test with Bonferroni correction. Detailed statistical information is provided in Online Methods.

  4. Radial sorting defects in Taz cKO-Yap cHet nerves are associated with a reduction in Schwann cell proliferation at P3.
    Figure 4: Radial sorting defects in Taz cKO–Yap cHet nerves are associated with a reduction in Schwann cell proliferation at P3.

    (a) Semithin cross-sections of sciatic nerves stained with Toluidine blue from control, Taz cKO and Taz cKO–Yap cHet mice at P3. Scale bar, 10 μm. n = 6 control, 4 Taz cKO and 3 Taz cKO–Yap cHet mice. (b) Numbers of myelinated fibers at P3 in control, Taz cKO and Taz cKO–Yap cHet mice. n = 6 control, 4 Taz cKO and 3 Taz cKO–Yap cHet mice. (c) TUNEL (red) and phosphorylated histone H3 (p-H3) staining (green) and DAPI (blue) analysis on longitudinal section of sciatic nerves from control and Taz cKO–Yap cHet at P3. Scale bar, 50 μm. n = 6 control, 4 Taz cKO and 3 Taz cKO–Yap cHet mice. (d) Relative numbers of TUNEL- and p-H3-positive nuclei and density and total number of nuclei in sciatic nerve (length of sciatic nerve measured = 400 μm). n = 3 mice per genotype. Each data point in b and d indicates the average value from one nerve from one mouse. Error bars indicate mean ± s.e.m. *P < 0.05, ***P < 0.001. Detailed statistical information is provided in Online Methods.

  5. Taz and Yap control expression of integrin [alpha]6 and dystroglycan in Schwann cells.
    Figure 5: Taz and Yap control expression of integrin α6 and dystroglycan in Schwann cells.

    (a) mRNA levels of Dag1 and Itga6 after verteporfin treatment (relative to DMSO-treated controls) in Schwann cells. n = 3 independent experiments with 3 independent samples per group. A logarithmic scale was used for the y axis, and the origin was set to 1. (b) mRNA expression (relative to wild-type mice) in Taz cKO and Taz cKO–Yap cHet cells at P20. n = 3 mice. A logarithmic scale was used for the y axis, and the origin was set to 1. (c) ChIP-qPCR analysis of Sox10 and Tead1 binding at Itga6 and Dag1 enhancer regions in P15 rat sciatic nerves. Binding was assessed relative to a negative control IP with goat IgG. The negative control is a region 17.8 kb from the Tekt3 gene, which is not expressed in Schwann cells. n = 3 independent experiments. (d,e) Western blot analysis (d (left) and e) and quantification (d, right) of integrins and β-dystroglycan (β-dyst.) expression in Taz cKO and Taz cKO–Yap cHet sciatic nerves at P20 (d) and P3 (e). Cnx, calnexin. For Dag1, n = 3 mice per group; for Itga6, n = 5 control and Taz cKO and 4 Taz cKO–Yap cHet mice; for Itgb4, n = 4 mice per group. (Full-length blots are presented in Supplementary Fig. 4.) (f–i) Localization of integrin α6 (f), β1 (g) and β4 subunits (h) and α-dystroglycan (i) in cross-sections of sciatic nerves at P20. Arrowheads indicate integrin α6 and β4 subunits (red) around the perineurium; arrows indicate integrin α6 and β4 subunits around Schwann cells (SC) in wild-type control nerves; 'ax' indicates axons. Error bars indicate mean ± s.d. (a,c) or mean ± s.e.m (b,d); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA with Bonferroni post hoc correction (a,c,d) or one-way ANOVA with Bonferroni post hoc correction (b). Scale bar, 10 μm. Detailed statistical information is provided in Online Methods.

  6. RNA-seq analysis of Taz cKO-Yap cHet sciatic, brachial and peripheral trigeminal nerves at P3.
    Figure 6: RNA-seq analysis of Taz cKO–Yap cHet sciatic, brachial and peripheral trigeminal nerves at P3.

    (a) Log2 fold change (FC) in Taz cKO–Yap cHet versus control mice plotted against the average count size for every gene. CPM, counts per million. Genes highlighted in red were significantly differentially expressed at 5% false discovery rate (FDR). Blue lines indicate twofold change. (b) Gene Ontology analysis of biological processes downregulated significantly in Taz cKO–Yap cHet mice. (c) Heatmap of the top 50 most significant differentially regulated genes. Scale indicates expression relative to median of six samples. (d) Heatmap of Srebf target genes (obtained from ref. 31). Scale indicates expression relative to median of six samples. All genes were downregulated in Taz cKO–Yap cHet mice.

  7. Laminin expression and basal lamina organization in Taz cKO and Taz cKO-Yap cHet nerves.
    Figure 7: Laminin expression and basal lamina organization in Taz cKO and Taz cKO–Yap cHet nerves.

    (a) Immunolocalization of laminin α2 chain (green) and neurofilament (NF; red) in cross sections of control, Taz cKO and Taz cKO–Yap cHet sciatic nerves at P20. Scale bar, 10 μm. n = 2 mice per genotype. (b) Western blot analysis of laminin (Lm) α2 and α5 chain expression at P20. n = 2 mice per genotype. (full-length blots are presented in Supplementary Fig. 4). (c) Electron micrographs of Taz cKO–Yap cHet sciatic nerves at P20. Arrowheads indicate basal lamina properly organized around amyelinated axons. n = 1 mouse per genotype. Scale bar, 500 nm. Cnx, calnexin.

  8. Schwann cell proliferation and apoptosis are not affected by Taz or Yap ablation at P20.
    Supplementary Fig. 1: Schwann cell proliferation and apoptosis are not affected by Taz or Yap ablation at P20.

    (a) TUNEL staining (red), pH3 staining (green) and DAPI (blue) analysis on longitudinal section of sciatic nerves from control, Taz and Yap mutants at P20. Scale bar, 100 µm. At least three animals per genotypes were analyzed. (b) Relative number of TUNEL and pH3 positive nuclei, density of nuclei (number of nuclei per mm2 of sciatic nerve) and total number of nuclei in sciatic nerve (length of sciatic nerve measured: 400 μm). The number of Schwann cells is decreased in double mutants. The increase of nuclei density in Taz cKO; Yap cHet and Yap/Taz cKO is likely caused by absence of myelin. n = 6 control mice, 3 Taz cKO, 4 Yap cKO, 4 Taz cKO Yap cHet, 5 Yap cKO Taz cHet; 3 Taz and Yap cKO. One way ANOVA TUNEL P = 0.4768 F (5, 15) = 0.9519 Taz cKO Yap cHet P = 0.1125; One way ANOVA nuclei per mm2 P < 0.0001, F (5, 18) = 18.54 with Bonferroni post hoc test Taz cKO P = 0.2804; Taz cKO Yap cHet P = 0.0002, Taz and Yap cKO Yap cHet P = 0.0062. Two-tailed unpaired Student's t test Taz and Yap cKO nuclei number P = 0.05. Error bars indicate s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

  9. The phenotypes of Yap and Taz cKO mice are not due to reduced expression of ErbB2, Cdc42, Egr2 or Sox10 in vivo.
    Supplementary Fig. 2: The phenotypes of Yap and Taz cKO mice are not due to reduced expression of ErbB2, Cdc42, Egr2 or Sox10 in vivo.

    (a) H3K27ac ChIP-Seq enrichment profiles in P15 rat sciatic nerve near (i) Erbb2, (ii) Cdc42, (iii) Egr2 and (iv) Sox10. H3K27ac regions were used to identify the presence of Tead motifs (vertical black bars). (b) Relative mRNA levels in primary rat Schwann cells treated with Verteporfin. Expression of ErbB2, Cdc42 and Sox10 are decreased upon Verteprofin treatment. Error bars indicate s.d. n = 3 independent experiments. Two way ANOVA P < 0.0001, F (2, 24) = 40.96 with Bonferroni post hoc test Erbb2 2 µM P = 0.0136, Erbb2 10 µM P < 0.0001, Cdc42 2 µM P = 0.2804, Cdc42 10 µM P < 0.0001, Egr2 2 µM P = 0.0134, Egr2 10 µM P < 0.0001, Sox10 2 µM P = 0.2312, Sox10 10 µM P < 0.0001. * P < 0.05, **** P < 0.0001. A logarithmic scale was used the y-axis and the origin was set to 1. (c-d) mRNA c) and protein d) levels in control, Taz cKO and Taz cKO; Yap cHet. Expression of Cdc42, ErbB2, Egr2 or Sox10 are not affected in vivo in the mutants. Error bars indicate s.e.m. n=3 (animal). n = 3 mice; One way ANOVA Cdc42 P = 0.3437, Dag1 F (2, 6) = 1.283 with Bonferroni post hoc test Taz cKO Yap cHet P = 0.3454; One way ANOVA ErbB2 P = 0.4162, F (2, 6) = 1.012 with Bonferroni post hoc test Taz cKO Yap cHet P = 0.476. A logarithmic scale was used the y-axis and the origin was set to 1. The western blots were cropped and the complete blots are presented in Supplementary Figure 4.

  10. Itga6 is regulated by Yap and Taz with Tead.
    Supplementary Fig. 3: Itga6 is regulated by Yap and Taz with Tead.

    (a) H3K27ac ChIP-Seq enrichment profiles in P15 rat sciatic nerve near (i) Itga6 and (ii) Dag1. H3K27ac regions were used to identify the presence of Tead motifs (vertical black bars). (b) ChIP-qPCR analysis on S16 Schwann cells. Enrichments are compared to goat IgG. The negative control site for ChIP-qPCR is a region 17.8 kb from the Tekt3 gene, which is not expressed in Schwann cells. Error bars indicate s.d. n=3 n = 3 independent experiments. Two way ANOVA P < 0.0001 F (2, 42) = 37.94 with Bonferroni post hoc test Sox10 – 20 kb P < 0.0001, Tead1 – 20 kb P < 0.0001, Sox10 – 7.6 kb P = 0.0026, Sox10 + 30 bp P < 0.0001. ** P < 0.01, **** P < 0.0001.

  11. Uncropped pictures of the Western blots shown in the manuscript.
    Supplementary Fig. 4: Uncropped pictures of the Western blots shown in the manuscript.
  12. Microscopy equipment and settings.
    Supplementary Fig. 5: Microscopy equipment and settings.

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Gene Expression Omnibus

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Author information

Affiliations

  1. Department of Biochemistry, Hunter James Kelly Research Institute, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York, USA.

    • Yannick Poitelon,
    • Kathleen Catignas,
    • Caterina Berti,
    • Marilena Palmisano,
    • Courtney Williamson,
    • Dominique Ameroso,
    • Kansho Abiko,
    • Yoonchan Hwang,
    • Lawrence Wrabetz &
    • Maria Laura Feltri

  2. Waisman Center, University of Wisconsin–Madison, Madison, Wisconsin, USA.

    • Camila Lopez-Anido &
    • John Svaren

  3. Lunenfeld–Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

    • Alex Gregorieff &
    • Jeffrey L Wrana

  4. Department of Biomedical Engineering, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York, USA.

    • Mohammadnabi Asmani &
    • Ruogang Zhao

  5. Department of Pharmacology and Toxicology, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York, USA.

    • Fraser James Sim

  6. Department of Neurology, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York, USA.

    • Lawrence Wrabetz &
    • Maria Laura Feltri

Contributions

Y.P., K.C., C.B., M.P. and M.L.F. designed research and interpreted data; Y.P. performed experiments with assistance from C.L.-A., K.C., C.B., M.P., C.W., D.A., K.A. and Y.H.; C.L.-A. and J.S. designed and performed ChIP sequencing and promoter analysis. M.A. and R.Z. designed and helped to perform biomechanical experiments; A.G. and J.L.W. and L.W. contributed analytical tools; F.J.S. analyzed RNA-seq data; Y.P. and M.L.F. wrote the manuscript; Y.P., C.L.-A., R.Z., F.J.S., J.S., L.W. and M.L.F. analyzed data and critically reviewed the manuscript.

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The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Schwann cell proliferation and apoptosis are not affected by Taz or Yap ablation at P20. (305 KB)

    (a) TUNEL staining (red), pH3 staining (green) and DAPI (blue) analysis on longitudinal section of sciatic nerves from control, Taz and Yap mutants at P20. Scale bar, 100 µm. At least three animals per genotypes were analyzed. (b) Relative number of TUNEL and pH3 positive nuclei, density of nuclei (number of nuclei per mm2 of sciatic nerve) and total number of nuclei in sciatic nerve (length of sciatic nerve measured: 400 μm). The number of Schwann cells is decreased in double mutants. The increase of nuclei density in Taz cKO; Yap cHet and Yap/Taz cKO is likely caused by absence of myelin. n = 6 control mice, 3 Taz cKO, 4 Yap cKO, 4 Taz cKO Yap cHet, 5 Yap cKO Taz cHet; 3 Taz and Yap cKO. One way ANOVA TUNEL P = 0.4768 F (5, 15) = 0.9519 Taz cKO Yap cHet P = 0.1125; One way ANOVA nuclei per mm2 P < 0.0001, F (5, 18) = 18.54 with Bonferroni post hoc test Taz cKO P = 0.2804; Taz cKO Yap cHet P = 0.0002, Taz and Yap cKO Yap cHet P = 0.0062. Two-tailed unpaired Student's t test Taz and Yap cKO nuclei number P = 0.05. Error bars indicate s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

  2. Supplementary Figure 2: The phenotypes of Yap and Taz cKO mice are not due to reduced expression of ErbB2, Cdc42, Egr2 or Sox10 in vivo. (523 KB)

    (a) H3K27ac ChIP-Seq enrichment profiles in P15 rat sciatic nerve near (i) Erbb2, (ii) Cdc42, (iii) Egr2 and (iv) Sox10. H3K27ac regions were used to identify the presence of Tead motifs (vertical black bars). (b) Relative mRNA levels in primary rat Schwann cells treated with Verteporfin. Expression of ErbB2, Cdc42 and Sox10 are decreased upon Verteprofin treatment. Error bars indicate s.d. n = 3 independent experiments. Two way ANOVA P < 0.0001, F (2, 24) = 40.96 with Bonferroni post hoc test Erbb2 2 µM P = 0.0136, Erbb2 10 µM P < 0.0001, Cdc42 2 µM P = 0.2804, Cdc42 10 µM P < 0.0001, Egr2 2 µM P = 0.0134, Egr2 10 µM P < 0.0001, Sox10 2 µM P = 0.2312, Sox10 10 µM P < 0.0001. * P < 0.05, **** P < 0.0001. A logarithmic scale was used the y-axis and the origin was set to 1. (c-d) mRNA c) and protein d) levels in control, Taz cKO and Taz cKO; Yap cHet. Expression of Cdc42, ErbB2, Egr2 or Sox10 are not affected in vivo in the mutants. Error bars indicate s.e.m. n=3 (animal). n = 3 mice; One way ANOVA Cdc42 P = 0.3437, Dag1 F (2, 6) = 1.283 with Bonferroni post hoc test Taz cKO Yap cHet P = 0.3454; One way ANOVA ErbB2 P = 0.4162, F (2, 6) = 1.012 with Bonferroni post hoc test Taz cKO Yap cHet P = 0.476. A logarithmic scale was used the y-axis and the origin was set to 1. The western blots were cropped and the complete blots are presented in Supplementary Figure 4.

  3. Supplementary Figure 3: Itga6 is regulated by Yap and Taz with Tead. (250 KB)

    (a) H3K27ac ChIP-Seq enrichment profiles in P15 rat sciatic nerve near (i) Itga6 and (ii) Dag1. H3K27ac regions were used to identify the presence of Tead motifs (vertical black bars). (b) ChIP-qPCR analysis on S16 Schwann cells. Enrichments are compared to goat IgG. The negative control site for ChIP-qPCR is a region 17.8 kb from the Tekt3 gene, which is not expressed in Schwann cells. Error bars indicate s.d. n=3 n = 3 independent experiments. Two way ANOVA P < 0.0001 F (2, 42) = 37.94 with Bonferroni post hoc test Sox10 – 20 kb P < 0.0001, Tead1 – 20 kb P < 0.0001, Sox10 – 7.6 kb P = 0.0026, Sox10 + 30 bp P < 0.0001. ** P < 0.01, **** P < 0.0001.

  4. Supplementary Figure 4: Uncropped pictures of the Western blots shown in the manuscript. (267 KB)
  5. Supplementary Figure 5: Microscopy equipment and settings. (534 KB)

PDF files

  1. Supplementary Text and Figures (1,302 KB)

    Supplementary Figures 1–5

  2. Supplementary Methods Checklist (428 KB)

Excel files

  1. Supplementary Table 1 (211 KB)

    Genes differentially expressed in Taz cKO–Yap cHet with a false discovery rate of 5% or lower.

Additional data