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

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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Figure 1: Yap and Taz expression and activation during Schwann cell development.
Figure 2: Laminins and mechanical stimulation regulate Yap and Taz in primary Schwann cells.
Figure 3: Ablation of Taz in Schwann cells impairs radial sorting of axons.
Figure 4: Radial sorting defects in Taz cKO–Yap cHet nerves are associated with a reduction in Schwann cell proliferation at P3.
Figure 5: Taz and Yap control expression of integrin α6 and dystroglycan in Schwann cells.
Figure 6: RNA-seq analysis of Taz cKO–Yap cHet sciatic, brachial and peripheral trigeminal nerves at P3.
Figure 7: Laminin expression and basal lamina organization in Taz cKO and Taz cKO–Yap cHet nerves.

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Acknowledgements

We thank E. Hurley for technical assistance; A. Sonnenberg (Netherlands Cancer Institute), D. Meijer and P. Brophy (Centre for Neuroregeneration, Edinburgh), the late G. Tarone (University of Turin), L. Sorokin (University of Muenster) and M. Wegner (Friedrich-Alexander University Erlangen–Nürnberg) for antibodies, and the late R. Quarles (National Institute of Neurological Diseases and Stroke) for the S16 cells. This work was funded by grants NS045630 (to M.L.F.), NS096104 (to L.W.) and NS075269 (to J.S.).

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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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Correspondence to Maria Laura Feltri.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 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.

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.

(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.

Supplementary Figure 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.

Supplementary Figure 4 Uncropped pictures of the Western blots shown in the manuscript.

Supplementary Figure 5 Microscopy equipment and settings.

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Supplementary Figures 1–5 (PDF 1271 kb)

Supplementary Methods Checklist (PDF 418 kb)

Supplementary Table 1

Genes differentially expressed in Taz cKO–Yap cHet with a false discovery rate of 5% or lower. (XLSX 206 kb)

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Poitelon, Y., Lopez-Anido, C., Catignas, K. et al. YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells. Nat Neurosci 19, 879–887 (2016). https://doi.org/10.1038/nn.4316

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