PMC

Persistence of Schwann cell proliferation. A–D, BrdU immunohistochemical analysis of P8 Chd4 cko sciatic nerve demonstrates an increased number of proliferating cells compared with control littermates. The BrdU-positive nuclei were counted, and proliferation was determined as a percentage of BrdU-positive nuclei compared with all Hoechst-stained nuclei in the nerve section. Scale bar, 30 μm. p < 0.019; n = 3 for each genotype.

ChIP analysis for occupancy of Egr2, Chd4, and MTA2 on Egr2 activated and repressed genes. The bar graph shows ChIP analysis of NuRD subunits, Chd4 and Mta2 (A), and Egr2 (B) binding to activated and repressed genes in P15 rat sciatic nerve. Quantitative PCR was used to calculate percentage recovery for both experimental and control (IgG) immunoprecipitations relative to total input, and the mean of three independent ChIP experiments is shown. The negative control site is an Egr2 negative binding site in Ndrg1. Error is shown as ±SEM. *p < 0.05.

NuRD complex components and Egr2 assemble on both activated and repressed genes during myelination. Chd4 binding is found on Sqle (A), and a negative regulator of myelination, c-Jun (B). ChIP-chip data for Egr2 () and two independent Chd4 antibodies were used to determine the binding location of Chd4 in P15 rat sciatic nerve. Data were analyzed using NimbleScan peak finding software. Peaks with a value of FDR <0.05 are colored in red. ChIP-chip analysis for occupancy of Egr2, Chd4, and MTA2 on the Egr2-repressed gene, c-Jun (B), confirms binding of these proteins to the same site upstream of the c-Jun promoter (box).

Chd4 deletion results in deregulation of Schwann cell genes induced and repressed during development. A, B, The mRNA expression levels for the indicated genes were determined by quantitative RT-PCR from sciatic nerve samples at the P8, P15, and P30 time points. Expression data are presented as a ratio of Chd4 mutant compared with wild-type mRNA levels (C). P30 protein levels of Egr2 (D), Oct6 (E), activated β-catenin (G), and P4 levels of Pmp22 (F) mirror the levels found in the mRNA expression data. Error bars indicate SD. Line represents relative level in wild-type mice. *p < 0.05; **p < 0.01; ***p < 0.005.

Ablation of Chd4 impairs the ability of Schwann cells to myelinate. Electron micrographs of sciatic nerve from control (A) and Chd4 cko (B) mice at P30. Magnification of myelin wraps show normal periodicity (C, inset). Scale bar, 0.1 μm. Scatter plot analysis of g ratio and axon diameter (D) shows the axon diameter is similar between genotypes; however, thinner myelin wraps axons of all diameters in Chd4 KO mice. Quantitation of the rate of dysmyelination indicates 24% of large-caliber axons contain abnormalities in myelination (E). Examples include amyelination, frayed myelin (F), or disorganized Schwann cell structures containing myelin debris (G). Scale bar: 1 and 2 μm, respectively. In addition, macrophage infiltration is characteristic of P30 nerve (H). Scale bar, 2 μm.

Chd4 mutant mice show a developmental delay. A, B, Electron microscopy analysis of sciatic nerve from wild-type and Chd4 mutant mice at P8, showing hypomyelination (arrows) and a radial sorting defect where large-caliber axons are still associated with Remak bundle axons (C, inset) (asterisks). Scale bars: 10 and 1 μm. D, The g ratio measures the ratio of axon diameter to its myelinated diameter. Unmyelinated Schwann cells are represented with a g ratio of 1 (box). Histogram error is given as mean SD for each bin. E, Axon classification into categories at P8: clustered-unmyelinated (dark gray), sorted-unmyelinated (light gray), or sorted-myelinated (black). P15 WT sciatic nerve (F) contrasts with hypomyelination (G, H) and radial sorting defects (I) in Chd4 mutant nerve. Scale bar, 2 μm.

Deficiencies in motor coordination and hindlimb reflex in Chd4 mutant mice. A, Immunohistochemistry of Chd4 (red) in the sciatic nerves of control and conditional knock-out mice at P8. Cell nuclei were counterstained with Hoechst (blue). Scale bar, 30 μm. B, Normal hindlimb postural reflex in a wild-type mouse (Chd4 f/f) characterized by spreading of the limbs. Abnormal reflex in Chd4 mutant mouse where hindlimbs are crossed and withdrawn (P55). C, Gross examination of sciatic nerve dissected from Chd4 KO and control mice at P30. D, RotaRod performance measuring latency to fall for 4-week-old mice. Three trials per session were measured with 20 min interval between trials. Statistical significance was determined using Student's t test: *p < 0.01; **p < 0.001. Error bars indicate SD.