Fig 2: Mecp2 -/y mice exhibit skeletal muscle disorganization and atrophy.(A) Representative pictures of TA and brain of 6 weeks old WT and Mecp2 -/y mice. Scale bars = 0,5 mm. (B) Measures of organ weights are indicated as means ± s.e.m. Unpaired t-test was performed and P values are shown. Table also reports the percentage of reduction observed in Mecp2 -/y mice (n≥6), calculated considering WT values as 100%. (C) Representative TA and gastrocnemius (Gast) muscle sections stained with H&E. Scale bar = 50 μm. (D) Representative TA and gastrocnemius muscle sections immunostained for Laminin. Scale bar = 50 μm. (E) CSA analysis of single muscle fibers, showing the abundance of smaller fibers in Mecp2 -/y mice compared to WT mice. At least 150 myofibers were counted for each animal/muscle, n = 3 per genotype. Left panel: quantification of mean CSA ± s.e.m. Significance is calculated with t test (**, P value: 0.0059 for TA; **, P value: 0.0089 for Gast). Central and right panels: size distribution of single muscle fibers. x-Axis = fiber size (μm2); y-Axis = percentage of fibers. (F) TA sections (n = 3) were stained with Sirius Red and the total amount of collagen was quantified with ImageJ (G); ns, P value: 0.5593. (H) Collagen I mRNA expression was assessed in WT and Mecp2 -/y gastrocnemius (n = 3). Significance is calculated with t test (ns, P value: 0.1126).

Fig 3: Muscle specific deletion of Mecp2 does not lead to the observed muscular alterations.(A) WBs confirm the specific skeletal muscle deletion of Mecp2 in the transgenic Mecp2 Flox MyoD iCre mice. WT, Mecp2 Flox and MyoD iCre genotypes are used as controls. (B) Representative TA cross-sections stained with Hematoxylin and Eosin (upper panels) or immunostained for Laminin (bottom panels). Scale bar = 50 μm. (C) Mean CSA ± s.e.m. showing no statistically significant differences between Mecp2 Flox MyoD iCre mice fibers and control ones. At least 130 myofibers were counted for each animal. Significance was calculated using one-way ANOVA (ns: 0.8520). (D) Mecp2 Flox MyoD iCre mice and controls show similar levels of Gast IGF-1 mRNA. Significance was calculated using one-way ANOVA (n≥3; ns: 0.9283). (E) Graph showing similar levels of P-rpS6 between Mecp2 Flox MyoD iCre quadriceps and the corresponding controls. Data are represented as mean ± s.e.m. (n = 2).

Fig 4: Overexpression of Irak1 in cortical neurons reduces dendritic arborization.(a) A plasmid overexpressing either a reporter GFP (Ctrl) or both Irak1 and GFP (Irak1) was transfected into developing immature neurons in culture. (b) IRAK1 protein (red) is highly expressed by neurons (MAP2 positive; blue) transfected with Irak1 and GFP (green), while neurons transfected with GFP alone express only endogenous, low levels of IRAK1. Large arrowheads indicate transfected neurons; small arrows indicate neighbouring, untransfected neurons. Scale bar, 50 μm. (c) Cells were immuno-labelled against both MAP2 and GFP, and the dendritic morphology of MAP2+/GFP+ cells was analysed by Sholl analysis. Overexpression of Irak1 significantly decreases neuronal dendritic arborization, compared to that of control neurons (control n=20, Irak1-overexpressing neurons n=36); two-way ANOVA, F(1,540)=39.9, P<0.001). Error bars=mean±s.e.m. (d) To investigate whether overexpression of Irak1 also modifies dendritic complexity of layer 2/3 neurons in vivo, Ctrl and Irak1 plasmids were injected in utero into the lateral ventricle (LV), and electroporated into neural precursors in the ventricular zone (VZ) at E15.5. Electroporated neural precursors subsequently give rise to differentiated cortical layer 2/3 projection neurons (NeuN+, red), with no obvious disruption in laminar location or survival at P14 with Irak1 overexpression. Scale bar, 200 μm. (e) IRAK1 protein (red) is highly overexpressed by P14 layer 2/3 neurons electroporated with Irak1, but not Ctrl, compared to endogenous IRAK1 expression within cortical layer 2/3 at P14. Scale bar, 50 μm. (f,g) We analysed the dendritic morphology of electroporated layer 2/3 projection neurons at P14 by Sholl analysis (ctrl n=24, Irak1 n=28). In vivo overexpression of Irak1 in layer 2/3 projection neurons results in reduced dendritic arborization, recapitulating the dendritic phenotype in layer 2/3 projection neurons in Mecp2-null mice. *P<0.01, a two-way ANOVA and the Bonferroni test, mean±s.e.m., Scale bars, 50 μm.

Fig 5: Methylation in striatal inhibitory neurons remains stable without MeCP2, but without Dnmt3a there is global loss of mCH and some loss of mCG.(A) Schematic of INTACT method used to isolate inhibitory neurons from the striata of WT, Dnmt3a cKO, or Mecp2 cKO mice that also conditionally express the INTACT allele. (B) Spearman correlation of methylation profiles from inhibitory neurons sorted from WT vs. Mecp2 cKO striatum, showing that methylation is stable in the absence of MeCP2. (C) Bar graph showing the global mCH level in each biological replicate (left). Example genes from DNA methylome sequencing tracks showing mCH signal in two biological replicates per genotype (right). (D) Bar graph showing the global mCG level in each biological replicate (left). Example genes from DNA methylome sequencing tracks showing mCG signal (right). Red bars indicate DMRs. (E) Genome wide correlation between mCH and mCG in wild-type mice showing poor correlation. (F) Genome wide correlation of Dnmt3a dependent mCG and mCH showing good correlation to indicate that mCH and mCG written by Dnmt3a are coupled. Correlation values for E and F are Pearson correlations designated as ‘rho’. n = 2 mice per genotype (see Materials and methods for specific genotype information). See Supplementary file 1 for replicate statistics.