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Zeb2 is essential for Schwann cell differentiation, myelination and nerve repair

Abstract

Schwann cell development and peripheral nerve myelination require the serial expression of transcriptional activators, such as Sox10, Oct6 (also called Scip or Pou3f1) and Krox20 (also called Egr2). Here we show that transcriptional repression, mediated by the zinc-finger protein Zeb2 (also known as Sip1), is essential for differentiation and myelination. Mice lacking Zeb2 in Schwann cells develop a severe peripheral neuropathy, caused by failure of axonal sorting and virtual absence of myelin membranes. Zeb2-deficient Schwann cells continuously express repressors of lineage progression. Moreover, genes for negative regulators of maturation such as Sox2 and Ednrb emerge as Zeb2 target genes, supporting its function as an 'inhibitor of inhibitors' in myelination control. When Zeb2 is deleted in adult mice, Schwann cells readily dedifferentiate following peripheral nerve injury and become repair cells. However, nerve regeneration and remyelination are both perturbed, demonstrating that Zeb2, although undetectable in adult Schwann cells, has a latent function throughout life.

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Figure 1: Functional analysis of Zeb2 in Schwann cell development and nerve repair.
Figure 2: Mice lacking Zeb2 in Schwann cells develop severe neuropathy.
Figure 3: Zeb2-deficient Schwann cells continuously express developmental inhibitors.
Figure 4: Zeb2-mediated repression of Ednrb and Hey2 is functionally relevant.
Figure 5: Zeb2 is required for efficient recovery after nerve injury.
Figure 6: Remyelination by Zeb2-deficient Schwann cells is impaired.
Figure 7: Dedifferentiation and redifferentiation of Zeb2-deficient Schwann cells

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Acknowledgements

The authors would like to thank C. Maack, T. Durkaya and A. Fahrenholz for excellent technical assistance. We thank the staff of the Transcriptome Analysis Laboratory (TAL) of the University Medical Center Göttingen for performance and statistical analysis of the microarray analysis and A. Diedrich, M. Wehe, B. Nickel and T. Hoffmeister for excellent support in animal husbandry. We are grateful to Q.R. Lu for communicating unpublished data. M.W.S. is supported by a Heisenberg Professorship granted by the DFG (GZ: SE 1944/1-1). D.H. is supported by Belspo-IAP funding (IAPVII-07), FWO-V (G.0782.14), Hercules Foundation (ZW09-03 project InfraMouse) and Erasmus MC start-up funds. K.-A.N. is supported by the DFG (Research Center Molecular Physiology of the Brain, CNMPB) and holds a European Research Council Advanced Investigator Grant.

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Authors and Affiliations

Authors

Contributions

S.Q. and B.G.B. designed the study, performed experiments and wrote the manuscript. M.E., T.K. and F.A.A. contributed to the experiments. F.F. performed luciferase assays. V.T., D.H., D.M. and U.S. provided transgenic mice. M.W. supervised F.F. and contributed to discussions. M.W.S. and K.-A.N. contributed to the manuscript and supervised the study.

Corresponding authors

Correspondence to Michael W Sereda or Klaus-Armin Nave.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Zeb2-deficient Schwann cells survive and exit the cell cycle normally.

(a) The number of Schwann cell nuclei per cross section of sciatic nerve was not different between conditional single mutants (Dhh-cre::Zeb2fl/fl) and both conditional double mutants (Dhh-cre::Zeb2fl/fl::Ednrbfl/fl and Dhh-cre::Zeb2fl/fl::Hey2fl/fl) at age P25. At age P365, the Schwann cell number of Dhh-cre::Zeb2fl/fl mice was not significantly different from P25. Bars, mean ±SD (n=3 animals per age and genotype, P=0.0522, H=7.717949, Kruskal-Wallis one-way ANOVA).

(b) Unaltered cell proliferation in sciatic nerve as determined by incorporation of bromo-desoxyuridine (BrdU) in Dhh-cre::Zeb2fl/fl mice compared to controls at E18.5, P10 and P25. Values are expressed as the percentage (±SD) of BrdU-positive cells of all DAPI-positive nuclei. (n=4 mice per genotype, except for the P10 time point n=3, two-sided Student’s t-test of unpaired samples E18.5 P=0.339, t=1.039350, P10 P=0.875, t=0.1679966, P25 P=0.697, t=0.4061983).

(c) Semi-thin sections of controls and mutants at age 1 year showing comparable size of sciatic nerve branches. Scale bar 50 µm.

(d) The number of axons counted in 15 randomly taken electron micrographs is not significantly different in mutants and controls. Bars, mean ±SD (n=3 animals per genotype, age 1 year, two-sided Student’s t-test of unpaired samples, P=0.192, t=1.568639).

Supplementary Figure 2 Zeb2 binding of target genes.

(a) In vivo chromatin immunoprecipitation of Zeb2 targets using DNA from pooled sciatic nerves of mice at age P1 (n=3 individual preparations of genomic DNA from 15 mice at age P1 in each preparation). Fold enrichment (by qPCR) of the indicated promoter fragments relative to control samples (no antibody) is shown for three independent experiments (variance introduced at the step of shearing pooled genomic DNA). A region in the E-cadherin promoter (Cdh, a known target gene of Zeb2) without Zeb2 binding site served as an independent negative control (neg). The location of the different primer pairs (#1 to #9) is shown schematically in (b).

Supplementary Figure 3 Confirmed Cre activity following tamoxifen-injection.

Analyzed 4 weeks after the last injection of tamoxifen, induced tdTomato fluorescence (red) was observed in cryostat cross sections of sciatic nerves from PLPCreERT2::Zeb2fl/fl mice (top panel), but not in tamoxifen-treated Zeb2fl/fl control mice (bottom panel). Green: myelin (fluoromyelin). Red: tdTomato. Scale bar, 10 µm (Representative images of n=3 mice per genoytpe).

Supplementary Figure 4 Zeb2 is dispensable for myelin maintenance in adult mice.

(a) Myelin sheath thickness was unaltered 12 weeks after the last injection of tamoxifen in PLP-creERT2::Zeb2fl/fl mice, when compared to the 3 respective control groups. Representative images of 3 animals per group. Scale bar, 2.5 µm.

(b) Scatter plot and g-ratio analysis of tamoxifen-treated PLP-creERT2::Zeb2fl/fl mice in comparison to tamoxifen-treated Zeb2fl/fl mice (n=3 animals per group, at least 100 randomly chosen fibers per animal, two-sided Student’s t-test of unpaired samples, P=0.504, t=0.7339136).

Supplementary Figure 5 Unaltered myelin sheath thickness after remyelination.

(a) Semi-thin sections of sciatic nerves from tamoxifen-treated Zeb2fl/fl and PLP-creERT2::Zeb2fl/fl mice 11 and 28 days after nerve crush. Note the reduced number of remyelinated fibers in nerves of PLP-creERT2::Zeb2fl/fl mice 28 days after injury (scale bar 20 µm).

(b) Scatter plot and G-ratio analysis of remyelinated fibers 8 weeks after nerve crush. Note that once remyelination occured resulting myelin sheath thickness was not significantly different in tamoxifen-treated PLP-creERT2::Zeb2fl/fl mice and the 3 respective control groups (n=3 animals per group, at least 100 randomly chosen fibers per animal, Kruskal-Wallis one-way ANOVA P=0.875, H=0.6923077).

Supplementary Figure 6 Hypothetical working model of Zeb2 function in the Schwann cell lineage.

Schwann cell (SC) differentiation is simplified in a linear order from SC precursors (left) to immature SC, to promyelin stage SC, and myelinating SC (right). Various promyelinating transcription factors drive differentiation (top: boxed in light green), whereas developmental inhibitors (bottom: boxed in light red) antagonize this process. In both groups, transcriptional activators (green fonts) and transcriptional repressors (red fonts) are found. Zeb2 is expressed throughout the early Schwann cell lineage and is a repressor of genes encoding developmental inhibitors ("inhibiting the inhibitors"), thereby promoting differentiation. Zeb2 inactivation in the Schwann cell lineage leads to continous expression of developmental inhibitors, such as Sox2, Hey2 and Ednrb (selected for analysis in this study), causing a developmental arrest of Zeb2-deficient SC prior to axonal sorting. When healthy adult nerves are injured, c-Jun drives SC dedifferentiation and the formation of 'repair cells', which is also possible in Zeb2 mutant mice. However, efficient redifferentiation to myelination-competent Schwann cells is Zeb2-dependent and often times fails in adult conditional Zeb2 mutants. For clarity, only a subset of known factors is depicted.

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Supplementary Figures 1–6 and Supplementary Table 1 (PDF 2104 kb)

Supplementary Methods Checklist (PDF 531 kb)

Conditional Zeb2 mutant at P25.

Dhh-cre::Zeb2fl/fl mouse at age P25 showing atactic movements and hind limb weakness (MPG 14786 kb)

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Quintes, S., Brinkmann, B., Ebert, M. et al. Zeb2 is essential for Schwann cell differentiation, myelination and nerve repair. Nat Neurosci 19, 1050–1059 (2016). https://doi.org/10.1038/nn.4321

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