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<t>MeCP2</t> dosage regulates heterochromatic structure in CA1 pyramidal cells. A, CA1 pyramidal cells of Mecp2+/− female mice (three months old) were stained with DAPI (blue) and MeCP2 (green). 3D structure of the foci was constructed and analyzed by Imaris (yellow). Note the change in the DAPI intensity (heat map) and in the shape of foci in MeCP2-negative (Null) cells when compared with the MeCP2-positive (WT) cells. Scale bars: 5 µm. B–F, Results of the quantification from the images obtained in A. B, DAPI intensity (int.) in the heterochromatic foci was higher in Null-cells than in WT-cells. B1, Relative DAPI intensity of each focus was normalized to the mean DAPI intensity of the foci in WT-cell. The graph shows cumulative distribution of the normalized mean DAPI intensity. N = 271 foci from 5 mice (WT-cells) and 238 foci from 5 mice (Null-cells). Kolmogorov–Smirnov test; p < 0.0001. B2, Bar graph showing mean DAPI intensity averaged in each mouse. Mean DAPI intensity was higher in Null-cells. N = 5 mice. Two-tailed paired t test; p = 0.0162, t(4) = 3.995. C, Ellipticity in the foci was decreased in Null-cells. C1, Cumulative distribution of ellipticity in the foci was shifted toward left in Null-cells. N = 271 foci from 5 mice (WT-cells) and 238 foci from 5 mice (Null-cells). Kolmogorov–Smirnov test; p < 0.0001. C2, Bar graph showing ellipticity averaged across foci in each mouse. Ellipticity was lower in Null-cells than WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.0008, t(4) = 9.137. D, Mean MeCP2 intensity was significantly reduced in Null-cells compared with WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.0004, t(4) = 11.13. E, Average volume of heterochromatic foci was comparable between Null-cells and WT-cells. N = 5 mice, Two-tailed paired t test; p = 0.1848, t(4) = 1.6. F, Average number of heterochromatic foci per cell was reduced in Null-cells compared with WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.0036, t(4) = 6.114. G, CA1 pyramidal cells of Mecp2Tg3/+ female mice (three months old) were stained and analyzed in the same way as in A. Scale bars: 5 µm. H–L, Quantified results from (G). Note the opposite changes in the parameters in Null-cells and Tg3-cells. H, DAPI intensity in the heterochromatic foci was reduced in Tg3-cells. H1, Relative DAPI intensity of each focus was normalized to the mean DAPI intensity of the foci in WT-cells. The graph shows cumulative distribution of the normalized mean DAPI intensity. N = 291 foci from 6 mice (WT-cells) and 256 foci from 6 mice (Tg3-cells). Kolmogorov–Smirnov test, p < 0.0001. H2, Bar graph showing mean DAPI intensity averaged in each mouse. Mean DAPI intensity was decreased in Tg3-cells. N = 6 mice. Two-tailed paired t test; p < 0.0001, t(5) = 16.92. I, Ellipticity in the foci was increased in Tg3-cells. I1, Cumulative distribution of ellipticity in each focus was shifted toward right in Tg3-cells. N = 291 foci from 6 mice (WT-cells) and 256 foci from 6 mice (Tg3-cells). Kolmogorov–Smirnov test; p < 0.0001. I2, Bar graph showing ellipticity averaged across foci in each mouse. Ellipticity was higher Tg3-cells than WT-cells. N = 6 mice. Two-tailed paired t test; p = 0.0422, t(5) = 2.711. J, Mean MeCP2 intensity (int.) was significantly higher in Tg3-cells than WT-cells. N = 6 mice. Two-tailed paired t test; p = 0.0037, t(5) = 5.114. K, Average volume of heterochromatic foci was comparable between Tg3-cells and WT-cells. N = 5 mice, Two-tailed paired t test; p = 0.2328, t(5) = 1.357. L, Average number of heterochromatic foci per cell was comparable between Tg3-cells and WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.1478, t(5) = 1.711. Bars show average ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, # p < 0.0001; n.s., not significant.
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MeCP2 dosage regulates heterochromatic structure in CA1 pyramidal cells. A, CA1 pyramidal cells of Mecp2+/− female mice (three months old) were stained with DAPI (blue) and MeCP2 (green). 3D structure of the foci was constructed and analyzed by Imaris (yellow). Note the change in the DAPI intensity (heat map) and in the shape of foci in MeCP2-negative (Null) cells when compared with the MeCP2-positive (WT) cells. Scale bars: 5 µm. B–F, Results of the quantification from the images obtained in A. B, DAPI intensity (int.) in the heterochromatic foci was higher in Null-cells than in WT-cells. B1, Relative DAPI intensity of each focus was normalized to the mean DAPI intensity of the foci in WT-cell. The graph shows cumulative distribution of the normalized mean DAPI intensity. N = 271 foci from 5 mice (WT-cells) and 238 foci from 5 mice (Null-cells). Kolmogorov–Smirnov test; p < 0.0001. B2, Bar graph showing mean DAPI intensity averaged in each mouse. Mean DAPI intensity was higher in Null-cells. N = 5 mice. Two-tailed paired t test; p = 0.0162, t(4) = 3.995. C, Ellipticity in the foci was decreased in Null-cells. C1, Cumulative distribution of ellipticity in the foci was shifted toward left in Null-cells. N = 271 foci from 5 mice (WT-cells) and 238 foci from 5 mice (Null-cells). Kolmogorov–Smirnov test; p < 0.0001. C2, Bar graph showing ellipticity averaged across foci in each mouse. Ellipticity was lower in Null-cells than WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.0008, t(4) = 9.137. D, Mean MeCP2 intensity was significantly reduced in Null-cells compared with WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.0004, t(4) = 11.13. E, Average volume of heterochromatic foci was comparable between Null-cells and WT-cells. N = 5 mice, Two-tailed paired t test; p = 0.1848, t(4) = 1.6. F, Average number of heterochromatic foci per cell was reduced in Null-cells compared with WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.0036, t(4) = 6.114. G, CA1 pyramidal cells of Mecp2Tg3/+ female mice (three months old) were stained and analyzed in the same way as in A. Scale bars: 5 µm. H–L, Quantified results from (G). Note the opposite changes in the parameters in Null-cells and Tg3-cells. H, DAPI intensity in the heterochromatic foci was reduced in Tg3-cells. H1, Relative DAPI intensity of each focus was normalized to the mean DAPI intensity of the foci in WT-cells. The graph shows cumulative distribution of the normalized mean DAPI intensity. N = 291 foci from 6 mice (WT-cells) and 256 foci from 6 mice (Tg3-cells). Kolmogorov–Smirnov test, p < 0.0001. H2, Bar graph showing mean DAPI intensity averaged in each mouse. Mean DAPI intensity was decreased in Tg3-cells. N = 6 mice. Two-tailed paired t test; p < 0.0001, t(5) = 16.92. I, Ellipticity in the foci was increased in Tg3-cells. I1, Cumulative distribution of ellipticity in each focus was shifted toward right in Tg3-cells. N = 291 foci from 6 mice (WT-cells) and 256 foci from 6 mice (Tg3-cells). Kolmogorov–Smirnov test; p < 0.0001. I2, Bar graph showing ellipticity averaged across foci in each mouse. Ellipticity was higher Tg3-cells than WT-cells. N = 6 mice. Two-tailed paired t test; p = 0.0422, t(5) = 2.711. J, Mean MeCP2 intensity (int.) was significantly higher in Tg3-cells than WT-cells. N = 6 mice. Two-tailed paired t test; p = 0.0037, t(5) = 5.114. K, Average volume of heterochromatic foci was comparable between Tg3-cells and WT-cells. N = 5 mice, Two-tailed paired t test; p = 0.2328, t(5) = 1.357. L, Average number of heterochromatic foci per cell was comparable between Tg3-cells and WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.1478, t(5) = 1.711. Bars show average ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, # p < 0.0001; n.s., not significant.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: MeCP2 dosage regulates heterochromatic structure in CA1 pyramidal cells. A, CA1 pyramidal cells of Mecp2+/− female mice (three months old) were stained with DAPI (blue) and MeCP2 (green). 3D structure of the foci was constructed and analyzed by Imaris (yellow). Note the change in the DAPI intensity (heat map) and in the shape of foci in MeCP2-negative (Null) cells when compared with the MeCP2-positive (WT) cells. Scale bars: 5 µm. B–F, Results of the quantification from the images obtained in A. B, DAPI intensity (int.) in the heterochromatic foci was higher in Null-cells than in WT-cells. B1, Relative DAPI intensity of each focus was normalized to the mean DAPI intensity of the foci in WT-cell. The graph shows cumulative distribution of the normalized mean DAPI intensity. N = 271 foci from 5 mice (WT-cells) and 238 foci from 5 mice (Null-cells). Kolmogorov–Smirnov test; p < 0.0001. B2, Bar graph showing mean DAPI intensity averaged in each mouse. Mean DAPI intensity was higher in Null-cells. N = 5 mice. Two-tailed paired t test; p = 0.0162, t(4) = 3.995. C, Ellipticity in the foci was decreased in Null-cells. C1, Cumulative distribution of ellipticity in the foci was shifted toward left in Null-cells. N = 271 foci from 5 mice (WT-cells) and 238 foci from 5 mice (Null-cells). Kolmogorov–Smirnov test; p < 0.0001. C2, Bar graph showing ellipticity averaged across foci in each mouse. Ellipticity was lower in Null-cells than WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.0008, t(4) = 9.137. D, Mean MeCP2 intensity was significantly reduced in Null-cells compared with WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.0004, t(4) = 11.13. E, Average volume of heterochromatic foci was comparable between Null-cells and WT-cells. N = 5 mice, Two-tailed paired t test; p = 0.1848, t(4) = 1.6. F, Average number of heterochromatic foci per cell was reduced in Null-cells compared with WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.0036, t(4) = 6.114. G, CA1 pyramidal cells of Mecp2Tg3/+ female mice (three months old) were stained and analyzed in the same way as in A. Scale bars: 5 µm. H–L, Quantified results from (G). Note the opposite changes in the parameters in Null-cells and Tg3-cells. H, DAPI intensity in the heterochromatic foci was reduced in Tg3-cells. H1, Relative DAPI intensity of each focus was normalized to the mean DAPI intensity of the foci in WT-cells. The graph shows cumulative distribution of the normalized mean DAPI intensity. N = 291 foci from 6 mice (WT-cells) and 256 foci from 6 mice (Tg3-cells). Kolmogorov–Smirnov test, p < 0.0001. H2, Bar graph showing mean DAPI intensity averaged in each mouse. Mean DAPI intensity was decreased in Tg3-cells. N = 6 mice. Two-tailed paired t test; p < 0.0001, t(5) = 16.92. I, Ellipticity in the foci was increased in Tg3-cells. I1, Cumulative distribution of ellipticity in each focus was shifted toward right in Tg3-cells. N = 291 foci from 6 mice (WT-cells) and 256 foci from 6 mice (Tg3-cells). Kolmogorov–Smirnov test; p < 0.0001. I2, Bar graph showing ellipticity averaged across foci in each mouse. Ellipticity was higher Tg3-cells than WT-cells. N = 6 mice. Two-tailed paired t test; p = 0.0422, t(5) = 2.711. J, Mean MeCP2 intensity (int.) was significantly higher in Tg3-cells than WT-cells. N = 6 mice. Two-tailed paired t test; p = 0.0037, t(5) = 5.114. K, Average volume of heterochromatic foci was comparable between Tg3-cells and WT-cells. N = 5 mice, Two-tailed paired t test; p = 0.2328, t(5) = 1.357. L, Average number of heterochromatic foci per cell was comparable between Tg3-cells and WT-cells. N = 5 mice. Two-tailed paired t test; p = 0.1478, t(5) = 1.711. Bars show average ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, # p < 0.0001; n.s., not significant.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Staining, Construct, Two Tailed Test

Impact of MeCP2 dosage on the heterochromatin structure of cortical excitatory cells. A, DAPI-foci in the cortical excitatory neurons of Mecp2+/− female mice (three months old). Neurons were stained for MeCP2 (green), DAPI (blue), and Camk2α (red). WT-cells and Null-cells were analyzed using the same method as in Figure 1. Scale bars: 5 µm. B–G, Quantified results from A. N = 6 mice. Two-tailed paired t test. B, Mean MeCP2 intensity (int.) was significantly reduced in Null-cells. p = 0.0032, t(5) = 5.299. C, Mean DAPI intensity was significantly increased in Null-cells. p = 0.0023, t(5) = 5.708. D, Maximum DAPI intensity within the heterochromatic foci was increased in Null-cells. p = 0.0016, t(5) = 6.157. E, Ellipticity of the heterochromatic foci was decreased in Null-cells. p = 0.0205, t(5) = 3.343. F, Average volume of foci was higher in Null-cells than in WT-cells. p = 0.0113, t(5) = 3.906. G, Average number of foci per nucleus was comparable between Null-cells and WT-cells. p = 0.0577, t(5) = 2.453. H, Heterochromatic foci in the cortical excitatory neurons of Mecp2Tg3/+ female mice (three months old). Neurons were stained for MeCP2 (green), DAPI (blue), and Camk2α (red). Scale bars: 5 µm. I–N, Quantified results from H. N = 6 mice. Two-tailed paired t test. I, Mean MeCP2 intensity was significantly increased in Tg3-cells. p = 0.0009, t(5) = 6.955. J, Mean DAPI intensity was significantly decreased in Tg3-cells. p = 0.0137, t(5) = 3.721. K, Maximum DAPI intensity within the heterochromatic foci was decreased in Tg3-cells. p = 0.0137, t(5) = 3.721. L, Ellipticity of the heterochromatic foci was increased in Tg3-cells. p = 0.0153, t(5) = 3.613. M, Average volume of foci was lower in Tg3-cells than in WT-cells. p = 0.0181, t(5) = 3.457. N, Average number of foci per nucleus was comparable between Tg3-cells and WT-cells. p = 0.5365, t(5) = 0.6632. Bars show average ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: Impact of MeCP2 dosage on the heterochromatin structure of cortical excitatory cells. A, DAPI-foci in the cortical excitatory neurons of Mecp2+/− female mice (three months old). Neurons were stained for MeCP2 (green), DAPI (blue), and Camk2α (red). WT-cells and Null-cells were analyzed using the same method as in Figure 1. Scale bars: 5 µm. B–G, Quantified results from A. N = 6 mice. Two-tailed paired t test. B, Mean MeCP2 intensity (int.) was significantly reduced in Null-cells. p = 0.0032, t(5) = 5.299. C, Mean DAPI intensity was significantly increased in Null-cells. p = 0.0023, t(5) = 5.708. D, Maximum DAPI intensity within the heterochromatic foci was increased in Null-cells. p = 0.0016, t(5) = 6.157. E, Ellipticity of the heterochromatic foci was decreased in Null-cells. p = 0.0205, t(5) = 3.343. F, Average volume of foci was higher in Null-cells than in WT-cells. p = 0.0113, t(5) = 3.906. G, Average number of foci per nucleus was comparable between Null-cells and WT-cells. p = 0.0577, t(5) = 2.453. H, Heterochromatic foci in the cortical excitatory neurons of Mecp2Tg3/+ female mice (three months old). Neurons were stained for MeCP2 (green), DAPI (blue), and Camk2α (red). Scale bars: 5 µm. I–N, Quantified results from H. N = 6 mice. Two-tailed paired t test. I, Mean MeCP2 intensity was significantly increased in Tg3-cells. p = 0.0009, t(5) = 6.955. J, Mean DAPI intensity was significantly decreased in Tg3-cells. p = 0.0137, t(5) = 3.721. K, Maximum DAPI intensity within the heterochromatic foci was decreased in Tg3-cells. p = 0.0137, t(5) = 3.721. L, Ellipticity of the heterochromatic foci was increased in Tg3-cells. p = 0.0153, t(5) = 3.613. M, Average volume of foci was lower in Tg3-cells than in WT-cells. p = 0.0181, t(5) = 3.457. N, Average number of foci per nucleus was comparable between Tg3-cells and WT-cells. p = 0.5365, t(5) = 0.6632. Bars show average ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Staining, Two Tailed Test

MeCP2-dependent structural changes of the heterochromatic foci in the cortical inhibitory neurons. A, DAPI-foci in the cortical PV-positive inhibitory neurons of Mecp2+/− female mice (three months old). Neurons were stained for MeCP2 (green), DAPI (blue), and PV (red). The images were analyzed using the same method as in Figures 1, ​,2.2. Scale bars: 5 µm. B–G, Quantified results from A. N = 6 mice. Two-tailed paired t test. B, Mean MeCP2 intensity (int.) was significantly reduced in Null-cells. p = 0.0006, t(5) = 7.771. C, Mean DAPI intensity was significantly increased in Null-cells. p = 0.0355, t(5) = 2.857. D, Maximum DAPI intensity within the heterochromatic foci was increased in Null-cells. p = 0.0474, t(5) = 2.615. E, Ellipticity of the heterochromatic foci was comparable between Null-cells and WT-cells. p = 0.1119, t(5) = 1.927. F, Average volume of foci was not changed in Null-cells. p = 0.9172, t(5) = 0.1093. G, Average number of foci per nucleus was comparable between Null-cells and WT-cells. p = 0.6920, t(5) = 0.42. H, DAPI-foci in the cortical PV-positive inhibitory neurons of Mecp2Tg3/ female mice (three months old). Scale bars: 5 µm. I–N, Quantified results from H. N = 6 mice. Two-tailed paired t test. I, Mean MeCP2 intensity was significantly higher in Tg3-cells. p = 0.0096, t(5) = 4.071. J, Mean DAPI intensity was significantly reduced in Tg3-cells. p = 0.0064, t(5) = 4.494. K, Maximum DAPI intensity within the heterochromatic foci was decreased in Tg3-cells. p = 0.0066, t(5) = 4.464. L, Ellipticity of the heterochromatic foci was comparable between Tg3-cells and WT-cells. p = 0.4481, t(5) = 0.8228. M, Average volume of foci was not changed in Tg3-cells. p = 0.5838, t(5) = 0.5853. N, Average number of foci per nucleus was comparable between Tg3-cells and WT-cells. p = 0.5219, t(5) = 0.6883. Bar graphs show average ± SEM; *p < 0.05, **p < 0.01, # p < 0.0001; n.s., not significant.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: MeCP2-dependent structural changes of the heterochromatic foci in the cortical inhibitory neurons. A, DAPI-foci in the cortical PV-positive inhibitory neurons of Mecp2+/− female mice (three months old). Neurons were stained for MeCP2 (green), DAPI (blue), and PV (red). The images were analyzed using the same method as in Figures 1, ​,2.2. Scale bars: 5 µm. B–G, Quantified results from A. N = 6 mice. Two-tailed paired t test. B, Mean MeCP2 intensity (int.) was significantly reduced in Null-cells. p = 0.0006, t(5) = 7.771. C, Mean DAPI intensity was significantly increased in Null-cells. p = 0.0355, t(5) = 2.857. D, Maximum DAPI intensity within the heterochromatic foci was increased in Null-cells. p = 0.0474, t(5) = 2.615. E, Ellipticity of the heterochromatic foci was comparable between Null-cells and WT-cells. p = 0.1119, t(5) = 1.927. F, Average volume of foci was not changed in Null-cells. p = 0.9172, t(5) = 0.1093. G, Average number of foci per nucleus was comparable between Null-cells and WT-cells. p = 0.6920, t(5) = 0.42. H, DAPI-foci in the cortical PV-positive inhibitory neurons of Mecp2Tg3/ female mice (three months old). Scale bars: 5 µm. I–N, Quantified results from H. N = 6 mice. Two-tailed paired t test. I, Mean MeCP2 intensity was significantly higher in Tg3-cells. p = 0.0096, t(5) = 4.071. J, Mean DAPI intensity was significantly reduced in Tg3-cells. p = 0.0064, t(5) = 4.494. K, Maximum DAPI intensity within the heterochromatic foci was decreased in Tg3-cells. p = 0.0066, t(5) = 4.464. L, Ellipticity of the heterochromatic foci was comparable between Tg3-cells and WT-cells. p = 0.4481, t(5) = 0.8228. M, Average volume of foci was not changed in Tg3-cells. p = 0.5838, t(5) = 0.5853. N, Average number of foci per nucleus was comparable between Tg3-cells and WT-cells. p = 0.5219, t(5) = 0.6883. Bar graphs show average ± SEM; *p < 0.05, **p < 0.01, # p < 0.0001; n.s., not significant.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Staining, Two Tailed Test

TEM analysis revealed subnuclear structures are altered in mice lacking and overexpressing MeCP2. A, TEM images of the nucleus in CA1 pyramidal cells obtained from Mecp2–/y and Mecp2+/y male mice (eight to nine weeks). Enlarged images show electron-dense foci. Scale bars: 2 µm (nucleus) and 1 µm (enlarged). B, C, Quantified result from A. B, Histogram showing the distribution for the irregularity of the electron-dense foci. The distribution was shifted toward left in Mecp2–/y mice. N = 3 mice. Two-way ANOVA (interaction), p = 0.0026, F(11,48) = 3.188. Bonferroni's multiple comparison test, p = 0.002 (irregularity = 1–1.5). C, Average irregularity of the electron-dense foci was significantly decreased in Mecp2–/y mice. N = 3 mice. Two-tailed t test, p < 0.0001, t(4) = 22.52. D, TEM images of the nucleus in CA1 pyramidal cells obtained from Mecp2Tg3/y and Mecp2+/y male mice (eight to nine weeks). Scale bars: 2 µm. E, F, Quantified results from E. E, Histogram showing the distribution of the irregularity of the electron-dense foci. The distribution was shifted toward right in Mecp2Tg3/y mice. N = 3 mice. Two-way ANOVA (interaction), p < 0.0001, F(11,44) = 5.535. Bonferroni's multiple comparison test, p = 0.0008 (irregularity = 1.0−1.5), 0.0029 (irregularity = 1.5–2.0), 0.0004 (irregularity > 6). F, Average irregularity of the electron-dense foci was significantly increased in Mecp2Tg3/y mice. N = 3 mice. Two-tailed t test, p = 0.0241, t(4) = 3.537. Bar graphs show average ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: TEM analysis revealed subnuclear structures are altered in mice lacking and overexpressing MeCP2. A, TEM images of the nucleus in CA1 pyramidal cells obtained from Mecp2–/y and Mecp2+/y male mice (eight to nine weeks). Enlarged images show electron-dense foci. Scale bars: 2 µm (nucleus) and 1 µm (enlarged). B, C, Quantified result from A. B, Histogram showing the distribution for the irregularity of the electron-dense foci. The distribution was shifted toward left in Mecp2–/y mice. N = 3 mice. Two-way ANOVA (interaction), p = 0.0026, F(11,48) = 3.188. Bonferroni's multiple comparison test, p = 0.002 (irregularity = 1–1.5). C, Average irregularity of the electron-dense foci was significantly decreased in Mecp2–/y mice. N = 3 mice. Two-tailed t test, p < 0.0001, t(4) = 22.52. D, TEM images of the nucleus in CA1 pyramidal cells obtained from Mecp2Tg3/y and Mecp2+/y male mice (eight to nine weeks). Scale bars: 2 µm. E, F, Quantified results from E. E, Histogram showing the distribution of the irregularity of the electron-dense foci. The distribution was shifted toward right in Mecp2Tg3/y mice. N = 3 mice. Two-way ANOVA (interaction), p < 0.0001, F(11,44) = 5.535. Bonferroni's multiple comparison test, p = 0.0008 (irregularity = 1.0−1.5), 0.0029 (irregularity = 1.5–2.0), 0.0004 (irregularity > 6). F, Average irregularity of the electron-dense foci was significantly increased in Mecp2Tg3/y mice. N = 3 mice. Two-tailed t test, p = 0.0241, t(4) = 3.537. Bar graphs show average ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Comparison, Two Tailed Test

Structural analysis of nucleoli in CA1 pyramidal cells and analysis of DAPI foci in the hepatocytes in MeCP2-deleted and MeCP2-overexpressing cells. A, CA1 pyramidal cells in Mecp2+/− female mice (three months old) were stained with RNA-selective dye (Syto RNASelect, green), MeCP2 (red), and DAPI (blue). Nucleoli were determined as Syto RNASelect-positive and DAPI-negative regions. Scale bars: 5 µm. B–D, Quantified data from A. N = 6 mice. Two-tailed paired t test. B, The irregularity of nucleoli was comparable between Null-cells and WT-cells. p = 0.3176. t(5) = 1.11. C, The number of nucleoli per cell was mildly decreased in Null-cells. p = 0.0046. t(5) = 1.11. D, Maximum area of nucleoli was comparable between Null-cells and WT-cells. p = 0.0767. t(5) = 2.225. E, Nucleoli of CA1 pyramidal cells in Mecp2Tg3/+ female mice (three months old) were visualized using the same method as in A. F–H, Quantified data from A. N = 6 mice. Two-tailed paired t test. F, The irregularity of nucleoli was comparable between Tg3-cells and WT-cells. p = 0.1312. t(5) = 1.803. G, The number of nucleoli per cell was comparable between Tg3-cells and WT-cells. p = 0.5155. t(5) = 0.6993. H, The maximum area of nucleoli was higher in Tg3-cells than WT-cells. p = 0.0390. t(5) = 2.779. I, Heterochromatic foci in the hepatocytes of Mecp2–/y and WT male mice (eight to nine weeks old). N = 5 (WT) and 4 (Mecp2–/y) mice. J, The number of foci per cell was not changed in Mecp2–/y mice. Mann–Whitney U test, p = 0.9058. K, Ellipticity was not changed in Mecp2–/y mice. Two-tailed t test, p = 0.6792, t(7) = 0.4313. L, Heterochromatic foci in the hepatocytes of Mecp2Tg3/y and WT male mice (eight to nine weeks old). N = 5 (WT) and N = 4 (Mecp2Tg3/y) mice. M, The number of foci per cell was not changed in Mecp2Tg3/y mice. Mann–Whitney U test, p = 0.5556. N, Ellipticity was not changed in Mecp2Tg3/y mice. Two-tailed t test, p = 0.6960, t(7) = 0.4072. Bar graphs show average ± SEM; *p < 0.05, **p < 0.01; n.s., not significant.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: Structural analysis of nucleoli in CA1 pyramidal cells and analysis of DAPI foci in the hepatocytes in MeCP2-deleted and MeCP2-overexpressing cells. A, CA1 pyramidal cells in Mecp2+/− female mice (three months old) were stained with RNA-selective dye (Syto RNASelect, green), MeCP2 (red), and DAPI (blue). Nucleoli were determined as Syto RNASelect-positive and DAPI-negative regions. Scale bars: 5 µm. B–D, Quantified data from A. N = 6 mice. Two-tailed paired t test. B, The irregularity of nucleoli was comparable between Null-cells and WT-cells. p = 0.3176. t(5) = 1.11. C, The number of nucleoli per cell was mildly decreased in Null-cells. p = 0.0046. t(5) = 1.11. D, Maximum area of nucleoli was comparable between Null-cells and WT-cells. p = 0.0767. t(5) = 2.225. E, Nucleoli of CA1 pyramidal cells in Mecp2Tg3/+ female mice (three months old) were visualized using the same method as in A. F–H, Quantified data from A. N = 6 mice. Two-tailed paired t test. F, The irregularity of nucleoli was comparable between Tg3-cells and WT-cells. p = 0.1312. t(5) = 1.803. G, The number of nucleoli per cell was comparable between Tg3-cells and WT-cells. p = 0.5155. t(5) = 0.6993. H, The maximum area of nucleoli was higher in Tg3-cells than WT-cells. p = 0.0390. t(5) = 2.779. I, Heterochromatic foci in the hepatocytes of Mecp2–/y and WT male mice (eight to nine weeks old). N = 5 (WT) and 4 (Mecp2–/y) mice. J, The number of foci per cell was not changed in Mecp2–/y mice. Mann–Whitney U test, p = 0.9058. K, Ellipticity was not changed in Mecp2–/y mice. Two-tailed t test, p = 0.6792, t(7) = 0.4313. L, Heterochromatic foci in the hepatocytes of Mecp2Tg3/y and WT male mice (eight to nine weeks old). N = 5 (WT) and N = 4 (Mecp2Tg3/y) mice. M, The number of foci per cell was not changed in Mecp2Tg3/y mice. Mann–Whitney U test, p = 0.5556. N, Ellipticity was not changed in Mecp2Tg3/y mice. Two-tailed t test, p = 0.6960, t(7) = 0.4072. Bar graphs show average ± SEM; *p < 0.05, **p < 0.01; n.s., not significant.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Staining, Two Tailed Test, MANN-WHITNEY

Structural changes in MeCP2-negative cells are present in presymptomatic young Mecp2+/− female mice. A, Representative images of the heterochromatic foci in CA1 pyramidal cells of a three-week-old Mecp2+/− female mouse. There was a mild but significant difference in the DAPI intensity and shape between Null-cells and WT-cells. Scale bars: 5 µm. B–F, Quantitative results from images taken in A. B1, Cumulative distribution of the mean DAPI intensity for each heterochromatic focus. DAPI intensity for each focus was normalized to the average DAPI intensity in the foci of the nearest WT-cells. The distribution for Null-cells was slightly shifted toward right. N = 362 foci (WT-cells) and 392 foci (Null-cells). Kolmogorov–Smirnov test, p = 0.0137. B2, Average of mean DAPI intensity from five mice was mildly increased in Null-cells. N = 5 mice. Two-tailed paired t test, p = 0.0023, t(4) = 6.93. C1, Cumulative distribution of ellipticity for each heterochromatic focus showed mild shift toward left in Null-cells. N = 362 foci (WT-cells) and 392 foci (Null-cells). Kolmogorov–Smirnov test, p = 0.00102. C2, Average ellipticity for five mice was significantly lower in Null-cells than WT-cells. N = 5 mice. Two-tailed paired t test, p = 0.0012, t(4) = 8.292. D, The intensity of MeCP2 was significantly decreased in Null-cells. N = 5 mice. Two-tailed paired t test, p = 0.0082, t(4) = 4.874. E, Average volume of foci was comparable between Null-cells and WT-cells. N = 5 mice. Two-tailed paired t test, p = 0.3344, t(4) = 1.097. F, The number of foci per cell was not different between Null-cells and WT-cells. N = 5 mice. Two-tailed paired t test, p = 0.1057, t(4) = 2.083. Bar graphs show average ± SEM; **p < 0.01; n.s., not significant.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: Structural changes in MeCP2-negative cells are present in presymptomatic young Mecp2+/− female mice. A, Representative images of the heterochromatic foci in CA1 pyramidal cells of a three-week-old Mecp2+/− female mouse. There was a mild but significant difference in the DAPI intensity and shape between Null-cells and WT-cells. Scale bars: 5 µm. B–F, Quantitative results from images taken in A. B1, Cumulative distribution of the mean DAPI intensity for each heterochromatic focus. DAPI intensity for each focus was normalized to the average DAPI intensity in the foci of the nearest WT-cells. The distribution for Null-cells was slightly shifted toward right. N = 362 foci (WT-cells) and 392 foci (Null-cells). Kolmogorov–Smirnov test, p = 0.0137. B2, Average of mean DAPI intensity from five mice was mildly increased in Null-cells. N = 5 mice. Two-tailed paired t test, p = 0.0023, t(4) = 6.93. C1, Cumulative distribution of ellipticity for each heterochromatic focus showed mild shift toward left in Null-cells. N = 362 foci (WT-cells) and 392 foci (Null-cells). Kolmogorov–Smirnov test, p = 0.00102. C2, Average ellipticity for five mice was significantly lower in Null-cells than WT-cells. N = 5 mice. Two-tailed paired t test, p = 0.0012, t(4) = 8.292. D, The intensity of MeCP2 was significantly decreased in Null-cells. N = 5 mice. Two-tailed paired t test, p = 0.0082, t(4) = 4.874. E, Average volume of foci was comparable between Null-cells and WT-cells. N = 5 mice. Two-tailed paired t test, p = 0.3344, t(4) = 1.097. F, The number of foci per cell was not different between Null-cells and WT-cells. N = 5 mice. Two-tailed paired t test, p = 0.1057, t(4) = 2.083. Bar graphs show average ± SEM; **p < 0.01; n.s., not significant.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Two Tailed Test

Levels of histone methylation marks in the heterochromatic foci of MeCP2-negative (Null) and MeCP2-overexpressing (Tg3) cells. Immunohistochemistry of CA1 pyramidal cells was performed using symptomatic Mecp2+/− (4–10 months old) and Mecp2Tg3/+ female mice (6–10 months old) to quantify H3K27me3 (A, E), H3K9me3 (B, F), H4K20me2/3 (C, G), and H3K4me3 (D, H) within the heterochromatic foci. Mean intensity of each histone modification mark was quantified by Imaris. The intensity was normalized to that of foci expressing normal level of MeCP2. MeCP2 deletion resulted in increased H3K27me3 (A) and H3K4me3 (D), while MeCP2 overexpression led to decreased H3K4me3 (H). H3K9me3 showed mild trend toward decrease in Null-cells and increase in Tg3-cells. Paired t test. N = 6 mice per genotype. A, H3K27me3 in Mecp2+/− mice. p = 0.0296, t(5) = 3.016. B, H3K9me3 in Mecp2+/− mice. p = 0.0926, t(5) = 2.075. C, H4K20me2/3 in Mecp2+/− mice. p = 0.4856, t(5) = 0.7525. D, H3K4me3 in Mecp2+/− mice. p = 0.0041, t(5) = 4.995. E, H3K27me3 in Mecp2Tg3/+ female mice. p = 0.8793, t(5) = 0.1598. F, H3K9me3 in Mecp2Tg3/+ mice. p = 0.0637, t(5) = 2.373. G, H4K20me2/3 in Mecp2Tg3/+ mice. p = 0.3484, t(5) = 1.034. H, H3K4me3 in Mecp2Tg3/+ mice. p = 0.0175, t(5) = 3.489. Arrowheads in the heatmaps indicate locations of the heterochromatic foci. Bar graphs show average ± SEM; *p < 0.05, **p < 0.01.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: Levels of histone methylation marks in the heterochromatic foci of MeCP2-negative (Null) and MeCP2-overexpressing (Tg3) cells. Immunohistochemistry of CA1 pyramidal cells was performed using symptomatic Mecp2+/− (4–10 months old) and Mecp2Tg3/+ female mice (6–10 months old) to quantify H3K27me3 (A, E), H3K9me3 (B, F), H4K20me2/3 (C, G), and H3K4me3 (D, H) within the heterochromatic foci. Mean intensity of each histone modification mark was quantified by Imaris. The intensity was normalized to that of foci expressing normal level of MeCP2. MeCP2 deletion resulted in increased H3K27me3 (A) and H3K4me3 (D), while MeCP2 overexpression led to decreased H3K4me3 (H). H3K9me3 showed mild trend toward decrease in Null-cells and increase in Tg3-cells. Paired t test. N = 6 mice per genotype. A, H3K27me3 in Mecp2+/− mice. p = 0.0296, t(5) = 3.016. B, H3K9me3 in Mecp2+/− mice. p = 0.0926, t(5) = 2.075. C, H4K20me2/3 in Mecp2+/− mice. p = 0.4856, t(5) = 0.7525. D, H3K4me3 in Mecp2+/− mice. p = 0.0041, t(5) = 4.995. E, H3K27me3 in Mecp2Tg3/+ female mice. p = 0.8793, t(5) = 0.1598. F, H3K9me3 in Mecp2Tg3/+ mice. p = 0.0637, t(5) = 2.373. G, H4K20me2/3 in Mecp2Tg3/+ mice. p = 0.3484, t(5) = 1.034. H, H3K4me3 in Mecp2Tg3/+ mice. p = 0.0175, t(5) = 3.489. Arrowheads in the heatmaps indicate locations of the heterochromatic foci. Bar graphs show average ± SEM; *p < 0.05, **p < 0.01.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Methylation, Immunohistochemistry, Modification, Expressing, Over Expression

AT-hook 2 domain of MeCP2 influences onset of heterochromatic structural changes in CA1 pyramidal cells. A, Representative images of heterochromatic foci in CA1 pyramidal cells obtained from eight- to nine-week-old WT (Mecp2+/y, WT), Mecp2–/y (Null), Mecp2–/y;MECP2-R270X (R270X), and Mecp2–/y; MECP2-G273X (G273X) mice. The cells were stained with anti-GFP to confirm expression of mutant MeCP2 which were tagged with GFP, and anti-MeCP2 C terminus antibody to confirm depletion of endogenous MeCP2. Scale bars: 5 µm. B, C, Quantitative results from the images in A. N = 7 (WT), N = 4 (Null), N = 4 (R270X), and N = 4 mice (G273X). One-way ANOVA followed by Tukey's multiple comparison test. B, Ellipticity of the heterochromatic foci was decreased in Mecp2-null mice and in mice expressing MeCP2-R270X, while it was not changed in mice expressing MeCP2-G273X. One-way ANOVA, p = 0.0002, F(3,15) = 13.27. Tukey's multiple comparison tests; p = 0.0013 (WT vs Null), 0.0003 (WT vs R270X), and 0.3267 (WT vs G273X). C, Average number of heterochromatic foci per cell was comparable in all the genotypes analyzed. One-way ANOVA, p = 0.1523, F(3,15) = 2.034. D, Heterochromatic foci in the CA1 pyramidal cells of five-month-old G273X and WT mice. E, Ellipticity of the heterochromatic foci was decreased in five-month-old G273X mice. N = 4 (WT) and N = 6 (G273X) mice. Two-tailed t test. p = 0.0026, t(8) = 4.298. F, Average number of heterochromatic foci per cell was not altered. N = 4 (WT) and N = 6 (G273X) mice. Mann–Whitney U test. p = 0.6619. Bar graphs show average ± SEM; **p < 0.01; n.s., not significant.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: AT-hook 2 domain of MeCP2 influences onset of heterochromatic structural changes in CA1 pyramidal cells. A, Representative images of heterochromatic foci in CA1 pyramidal cells obtained from eight- to nine-week-old WT (Mecp2+/y, WT), Mecp2–/y (Null), Mecp2–/y;MECP2-R270X (R270X), and Mecp2–/y; MECP2-G273X (G273X) mice. The cells were stained with anti-GFP to confirm expression of mutant MeCP2 which were tagged with GFP, and anti-MeCP2 C terminus antibody to confirm depletion of endogenous MeCP2. Scale bars: 5 µm. B, C, Quantitative results from the images in A. N = 7 (WT), N = 4 (Null), N = 4 (R270X), and N = 4 mice (G273X). One-way ANOVA followed by Tukey's multiple comparison test. B, Ellipticity of the heterochromatic foci was decreased in Mecp2-null mice and in mice expressing MeCP2-R270X, while it was not changed in mice expressing MeCP2-G273X. One-way ANOVA, p = 0.0002, F(3,15) = 13.27. Tukey's multiple comparison tests; p = 0.0013 (WT vs Null), 0.0003 (WT vs R270X), and 0.3267 (WT vs G273X). C, Average number of heterochromatic foci per cell was comparable in all the genotypes analyzed. One-way ANOVA, p = 0.1523, F(3,15) = 2.034. D, Heterochromatic foci in the CA1 pyramidal cells of five-month-old G273X and WT mice. E, Ellipticity of the heterochromatic foci was decreased in five-month-old G273X mice. N = 4 (WT) and N = 6 (G273X) mice. Two-tailed t test. p = 0.0026, t(8) = 4.298. F, Average number of heterochromatic foci per cell was not altered. N = 4 (WT) and N = 6 (G273X) mice. Mann–Whitney U test. p = 0.6619. Bar graphs show average ± SEM; **p < 0.01; n.s., not significant.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Staining, Expressing, Mutagenesis, Comparison, Two Tailed Test, MANN-WHITNEY

Knock-down of MeCP2 in the adult cortical pyramidal cells induces reduction in the number of heterochromatic foci. A, AAV(DJ)-vector inducing expression of Mecp2-targetting miRNA and GFP (AAV-mirMecp2) was injected into the PFC of WT mice (six to seven weeks). The mice were perfused five weeks after the injection, and the brain slices were stained with DAPI and antibodies for MeCP2, GFP, and Camk2α to mark pyramidal cells. Images are single plane images showing GFP and Camk2α signals (left panel) and 3D-reconstructed images from the same cells (right panels). Scale bars: 5 µm. B–F, Results from the quantitative analysis of DAPI foci. Reduction of MeCP2 was confirmed in GFP-positive cells (B, p = 0.0024, t(4) = 6.815). Average number of foci within the nucleus was decreased by Mecp2 knock-down (F, p = 0.0085, t(4) = 4.832). No changes were detected in the mean DAPI intensity (C, p = 0.6623, t(4) = 0.4708), ellipticity (D, p = 0.6471, t(4) = 0.4942), or volume (E, p = 0.1037, t(4) = 2.099). Paired t test. N = 5 injected area from 3 mice. G, AAV(DJ)-vector inducing expression of random sequence miRNA and GFP (AAV-mirNega) was injected into the PFC of WT mice (six to seven weeks), and the analysis was done in the same way as in A–F. H–L, Results from the quantitative analysis of DAPI foci. No change was detected in MeCP2 intensity (H, p = 0.6486, t(4) = 0.4919), DAPI intensity (I, p = 0.1705, t(4) = 1.669), ellipticity (J, p = 0.99, t(4) = 0.00018), number of foci (K, p = 0.8717, t(4) = 0.1722), or volume (L, p = 0.7768, t(4) = 0.3717). Paired t test. N = 5 injected area from 3 mice. Bar graphs show average ± SEM; **p < 0.01.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: Knock-down of MeCP2 in the adult cortical pyramidal cells induces reduction in the number of heterochromatic foci. A, AAV(DJ)-vector inducing expression of Mecp2-targetting miRNA and GFP (AAV-mirMecp2) was injected into the PFC of WT mice (six to seven weeks). The mice were perfused five weeks after the injection, and the brain slices were stained with DAPI and antibodies for MeCP2, GFP, and Camk2α to mark pyramidal cells. Images are single plane images showing GFP and Camk2α signals (left panel) and 3D-reconstructed images from the same cells (right panels). Scale bars: 5 µm. B–F, Results from the quantitative analysis of DAPI foci. Reduction of MeCP2 was confirmed in GFP-positive cells (B, p = 0.0024, t(4) = 6.815). Average number of foci within the nucleus was decreased by Mecp2 knock-down (F, p = 0.0085, t(4) = 4.832). No changes were detected in the mean DAPI intensity (C, p = 0.6623, t(4) = 0.4708), ellipticity (D, p = 0.6471, t(4) = 0.4942), or volume (E, p = 0.1037, t(4) = 2.099). Paired t test. N = 5 injected area from 3 mice. G, AAV(DJ)-vector inducing expression of random sequence miRNA and GFP (AAV-mirNega) was injected into the PFC of WT mice (six to seven weeks), and the analysis was done in the same way as in A–F. H–L, Results from the quantitative analysis of DAPI foci. No change was detected in MeCP2 intensity (H, p = 0.6486, t(4) = 0.4919), DAPI intensity (I, p = 0.1705, t(4) = 1.669), ellipticity (J, p = 0.99, t(4) = 0.00018), number of foci (K, p = 0.8717, t(4) = 0.1722), or volume (L, p = 0.7768, t(4) = 0.3717). Paired t test. N = 5 injected area from 3 mice. Bar graphs show average ± SEM; **p < 0.01.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Knockdown, Plasmid Preparation, Expressing, Injection, Staining, Sequencing

AAV-induced overexpression of MeCP2 caused heterochromatin changes in the adult cortical pyramidal cells. A, AAV(DJ)-DIO-huMECP2-GFP and AAV5-Camk2α-Cre were co-injected into the PFC of mature WT mice (six to seven weeks), and the brains were analyzed three weeks after the injection. The brain slices were stained with DAPI and antibodies against MeCP2 and GFP. The cells were grouped into GFP-negative, GFP-low, and GFP-high group based on the GFP intensity. Scale bars: 5 µm. B–G, Results from the quantitative analysis of DAPI foci. N = 6 injected sites from 3 mice. B, MeCP2 intensity was significantly increased with higher GFP expression. Friedman's ANOVA, p = 0.001; Dunn's multiple comparison test, p = 0.0011 (GFP-negative vs GFP-high). C, DAPI intensity within the foci was decreased in GFP-low and GFP-high cells. Friedman's ANOVA, p = 0.0055; Dunn's multiple comparison test, p = 0.0418 (GFP-negative vs GFP-low), p = 0.0078 (GFP-negative vs GFP-high). D, Ellipticity was not affected by MeCP2 expression. One-way ANOVA, p = 0.3881. E, Mean volume was not affected by MeCP2 overexpression. Friedman's ANOVA, p = 0.2522. F, Maximum volume of foci within each nucleus was significantly increased in GFP-high group. Friedman's ANOVA, p = 0.0001; Dunn's multiple comparison test, p = 0.1665 (GFP-negative vs GFP-low) and p = 0.0011 (GFP-negative vs GFP-high). G, Average number of foci was not changed. Friedman's ANOVA, p = 0.1416. Bar graphs show average ± SEM; *p < 0.05. **p < 0.01.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: AAV-induced overexpression of MeCP2 caused heterochromatin changes in the adult cortical pyramidal cells. A, AAV(DJ)-DIO-huMECP2-GFP and AAV5-Camk2α-Cre were co-injected into the PFC of mature WT mice (six to seven weeks), and the brains were analyzed three weeks after the injection. The brain slices were stained with DAPI and antibodies against MeCP2 and GFP. The cells were grouped into GFP-negative, GFP-low, and GFP-high group based on the GFP intensity. Scale bars: 5 µm. B–G, Results from the quantitative analysis of DAPI foci. N = 6 injected sites from 3 mice. B, MeCP2 intensity was significantly increased with higher GFP expression. Friedman's ANOVA, p = 0.001; Dunn's multiple comparison test, p = 0.0011 (GFP-negative vs GFP-high). C, DAPI intensity within the foci was decreased in GFP-low and GFP-high cells. Friedman's ANOVA, p = 0.0055; Dunn's multiple comparison test, p = 0.0418 (GFP-negative vs GFP-low), p = 0.0078 (GFP-negative vs GFP-high). D, Ellipticity was not affected by MeCP2 expression. One-way ANOVA, p = 0.3881. E, Mean volume was not affected by MeCP2 overexpression. Friedman's ANOVA, p = 0.2522. F, Maximum volume of foci within each nucleus was significantly increased in GFP-high group. Friedman's ANOVA, p = 0.0001; Dunn's multiple comparison test, p = 0.1665 (GFP-negative vs GFP-low) and p = 0.0011 (GFP-negative vs GFP-high). G, Average number of foci was not changed. Friedman's ANOVA, p = 0.1416. Bar graphs show average ± SEM; *p < 0.05. **p < 0.01.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Over Expression, Injection, Staining, Expressing, Comparison

MeCP2 overexpression in the adult PFC induces altered response to observational fear. A, Experimental design. AAVs were injected into the bilateral PFC of WT mice at six to seven weeks. AAV5-Camk2α-Cre and AAV(DJ)-DIO-huMECP2-GFP were used to induce overexpression of MeCP2 in the cortical pyramidal cells (Cre:MeCP2-GFP). Control mice received injection of either AAV5-Camk2α-Cre only (Cre) or AAV5-Camk2α-Cre together with AAV(DJ)-DIO-GFP (Cre:GFP). LD test and OF activity test were performed two and three weeks after the injection. Observational fear response (OBS) was performed at 3.5 weeks. B, Images of coronal slices showing distribution of GFP around injection areas. PL, prelimbic. IL, infralimbic. Scale bars: 1 mm. C, Results from OF and LD tests two weeks after the injection. No change was found. One-way ANOVA. p = 0.5881, F(2,30) = 0.5403 (OF, total distance), p = 0.8259, F(2,30) = 0.1925 (OF, center time), p = 0.586, F(2,24) = 0.5465 (LD). N (Cre, Cre: MeCP2-GFP, Cre:GFP) = (11, 10, 12) and (9, 8, 10) mice for OF and LD, respectively. D, Results from OF and LD tests three weeks after the injection. Mice overexpressing MeCP2 (Cre: MeCP2-GFP) showed trend toward increased time in light in the LD test. One-way ANOVA. p = 0.4695, F(2,31) = 0.7749 (OF, total distance), p = 0.2468, F(2,31) = 1.464 (OF, center time), p = 0.0475, F(2,31) = 3.367 (LD). Tukey's multiple comparison test for LD, p = 0.0583 (Cre:MeCP2-GFP vs Cre:GFP) and p = 0.1085 (Cre vs Cre:MeCP2-GFP). N (Cre, Cre:MeCP2-GFP, Cre:GFP) = (11, 11, 12) mice for OF and LD. E, Experimental design for the OBS test. See method section for details. F, Results from OBS test. Mice overexpressing MeCP2 (Cre:MeCP2-GFP) responded to the observational fear with higher freezing time in the after-shock phase. There was no significant change in the freezing time at the baseline and during the shock phase. One-way ANOVA. p = 0.4203, F(2,29) = 0.8931 (baseline); p = 0.0521, F(2,29) = 3.278 (during shock); p = 0.0260, F(2,29) = 4.150 (after shock). Tukey's multiple comparison test for after shock, p = 0.0295 (Cre:MeCP2-GFP vs Cre:GFP), p = 0.0784 (Cre vs Cre:MeCP2-GFP), p = 0.9355 (Cre vs Cre:GFP). N = 10 (Cre), N = 10 (Cre:MeCP2-GFP), and N = 12 (Cre:GFP) mice. Bar graphs show average ± SEM; *p < 0.05.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: MeCP2 overexpression in the adult PFC induces altered response to observational fear. A, Experimental design. AAVs were injected into the bilateral PFC of WT mice at six to seven weeks. AAV5-Camk2α-Cre and AAV(DJ)-DIO-huMECP2-GFP were used to induce overexpression of MeCP2 in the cortical pyramidal cells (Cre:MeCP2-GFP). Control mice received injection of either AAV5-Camk2α-Cre only (Cre) or AAV5-Camk2α-Cre together with AAV(DJ)-DIO-GFP (Cre:GFP). LD test and OF activity test were performed two and three weeks after the injection. Observational fear response (OBS) was performed at 3.5 weeks. B, Images of coronal slices showing distribution of GFP around injection areas. PL, prelimbic. IL, infralimbic. Scale bars: 1 mm. C, Results from OF and LD tests two weeks after the injection. No change was found. One-way ANOVA. p = 0.5881, F(2,30) = 0.5403 (OF, total distance), p = 0.8259, F(2,30) = 0.1925 (OF, center time), p = 0.586, F(2,24) = 0.5465 (LD). N (Cre, Cre: MeCP2-GFP, Cre:GFP) = (11, 10, 12) and (9, 8, 10) mice for OF and LD, respectively. D, Results from OF and LD tests three weeks after the injection. Mice overexpressing MeCP2 (Cre: MeCP2-GFP) showed trend toward increased time in light in the LD test. One-way ANOVA. p = 0.4695, F(2,31) = 0.7749 (OF, total distance), p = 0.2468, F(2,31) = 1.464 (OF, center time), p = 0.0475, F(2,31) = 3.367 (LD). Tukey's multiple comparison test for LD, p = 0.0583 (Cre:MeCP2-GFP vs Cre:GFP) and p = 0.1085 (Cre vs Cre:MeCP2-GFP). N (Cre, Cre:MeCP2-GFP, Cre:GFP) = (11, 11, 12) mice for OF and LD. E, Experimental design for the OBS test. See method section for details. F, Results from OBS test. Mice overexpressing MeCP2 (Cre:MeCP2-GFP) responded to the observational fear with higher freezing time in the after-shock phase. There was no significant change in the freezing time at the baseline and during the shock phase. One-way ANOVA. p = 0.4203, F(2,29) = 0.8931 (baseline); p = 0.0521, F(2,29) = 3.278 (during shock); p = 0.0260, F(2,29) = 4.150 (after shock). Tukey's multiple comparison test for after shock, p = 0.0295 (Cre:MeCP2-GFP vs Cre:GFP), p = 0.0784 (Cre vs Cre:MeCP2-GFP), p = 0.9355 (Cre vs Cre:GFP). N = 10 (Cre), N = 10 (Cre:MeCP2-GFP), and N = 12 (Cre:GFP) mice. Bar graphs show average ± SEM; *p < 0.05.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Over Expression, Injection, Control, Activity Assay, Comparison

MeCP2 overexpression in the adult cortical neurons induces MeCP2 dosage-dependent transcriptional changes. A, Experimental design. AAV5-Camk2α-Cre and AAV(DJ)-DIO-huMECP2-GFP were used to induce overexpression of MeCP2 in the bilateral PFC of adult mice (six to seven weeks). PFC was dissected three weeks after the injection and neuronal nuclei labeled by anti-NeuN antibody were processed by FACS. The nuclei were sorted into four groups based on the GFP intensity and used for mRNA-Seq. Results from mRNA-Seq analysis including the number of mapped reads and the list of DEGs are summarized in Extended Data Figure 12-1. B, Images of the sorted nuclei. Scale bars: 5 µm. C–I, Results from mRNA-Seq analysis. DEGs were obtained using GFP-negative group as a control. C, Transcript levels of endogenous mouse Mecp2 (left), exogenous human MECP2 (middle), and total Mecp2 normalized to the control. Overexpression of human MECP2 did not cause significant changes in the endogenous mouse Mecp2 (left). Total Mecp2 reads correlated positively with the GFP expression levels. D, Number of DEGs in GFP-low, GFP-med, and GFP-high groups. Higher numbers of DEGs were detected with higher expression of MECP2. E, Heatmap showing log2FC for genes altered in both GFP-med and GFP-high groups. Absolute log2FC in GFP-high group was consistently higher than that in GFP-med group. F, G, Difference in the absolute log2FC between GFP-med and GFP-high groups were plotted in y-axis and the absolute log2FC for each gene in GFP-med group were plotted in x-axis. Values in y-axis were higher than zero in the majority of common DEGs in both upregulated genes (F) and downregulated genes (G). The fractions of these genes are indicated in red. H, I, Comparison of log2FC for common DEGs between GFP-med and GFP-high groups. The analysis was done in both upregulated genes (H) and downregulated genes (I). The results show that the absolute log2FC for each gene is consistently higher in GFP-high group than in GFP-med group. Wilcoxon matched pair signed rank test. p < 0.0001, N = 103 genes (H); p < 0.0001, N = 108 genes (I). J, Results from RT-qPCR for the subset of common DEGs with the most significant FDRs. Fold change was obtained by normalizing Ct of each gene with that of Gapdh. The magnitude of fold change was consistently higher in GFP-high group and clearly correlated with MECP2 expression levels. One-way ANOVA: p < 0.0001, F(2.427,7.281) = 106.1 (Gipr), p = 0.0011, F(1.971,5.912) = 27.24 (Efnb3), p = 0.0031, F(1.299,3.897) = 39.50 (Pcdh9), p = 0.0025, F(1.265,3.796) = 47.24 (Slc24a2). ANOVA was followed by post hoc Tukey's multiple comparison tests. Gipr: p = 0.0121 (negative vs med), p = 0.0036 (negative vs high), p = 0.0128 (low vs med), p = 0.0021 (low vs high), p = 0.0067 (med vs high). Efnb3: p = 0.0075 (negative vs high) and 0.0164 (low vs high). Pcdh9: p = 0.0239 (negative vs low), 0.0021 (negative vs med), and 0.0074 (negative vs high). Slc24a2: p = 0.0427 (negative vs low), p = 0.0014 (negative vs med), p = 0.0099 (negative vs high), p = 0.0016 (low vs med), and p = 0.0309 (low vs high). Bar graphs show average ± SEM; *p < 0.05, **p < 0.001, ****p < 0.0001.

Journal: The Journal of Neuroscience

Article Title: MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

doi: 10.1523/JNEUROSCI.1281-19.2020

Figure Lengend Snippet: MeCP2 overexpression in the adult cortical neurons induces MeCP2 dosage-dependent transcriptional changes. A, Experimental design. AAV5-Camk2α-Cre and AAV(DJ)-DIO-huMECP2-GFP were used to induce overexpression of MeCP2 in the bilateral PFC of adult mice (six to seven weeks). PFC was dissected three weeks after the injection and neuronal nuclei labeled by anti-NeuN antibody were processed by FACS. The nuclei were sorted into four groups based on the GFP intensity and used for mRNA-Seq. Results from mRNA-Seq analysis including the number of mapped reads and the list of DEGs are summarized in Extended Data Figure 12-1. B, Images of the sorted nuclei. Scale bars: 5 µm. C–I, Results from mRNA-Seq analysis. DEGs were obtained using GFP-negative group as a control. C, Transcript levels of endogenous mouse Mecp2 (left), exogenous human MECP2 (middle), and total Mecp2 normalized to the control. Overexpression of human MECP2 did not cause significant changes in the endogenous mouse Mecp2 (left). Total Mecp2 reads correlated positively with the GFP expression levels. D, Number of DEGs in GFP-low, GFP-med, and GFP-high groups. Higher numbers of DEGs were detected with higher expression of MECP2. E, Heatmap showing log2FC for genes altered in both GFP-med and GFP-high groups. Absolute log2FC in GFP-high group was consistently higher than that in GFP-med group. F, G, Difference in the absolute log2FC between GFP-med and GFP-high groups were plotted in y-axis and the absolute log2FC for each gene in GFP-med group were plotted in x-axis. Values in y-axis were higher than zero in the majority of common DEGs in both upregulated genes (F) and downregulated genes (G). The fractions of these genes are indicated in red. H, I, Comparison of log2FC for common DEGs between GFP-med and GFP-high groups. The analysis was done in both upregulated genes (H) and downregulated genes (I). The results show that the absolute log2FC for each gene is consistently higher in GFP-high group than in GFP-med group. Wilcoxon matched pair signed rank test. p < 0.0001, N = 103 genes (H); p < 0.0001, N = 108 genes (I). J, Results from RT-qPCR for the subset of common DEGs with the most significant FDRs. Fold change was obtained by normalizing Ct of each gene with that of Gapdh. The magnitude of fold change was consistently higher in GFP-high group and clearly correlated with MECP2 expression levels. One-way ANOVA: p < 0.0001, F(2.427,7.281) = 106.1 (Gipr), p = 0.0011, F(1.971,5.912) = 27.24 (Efnb3), p = 0.0031, F(1.299,3.897) = 39.50 (Pcdh9), p = 0.0025, F(1.265,3.796) = 47.24 (Slc24a2). ANOVA was followed by post hoc Tukey's multiple comparison tests. Gipr: p = 0.0121 (negative vs med), p = 0.0036 (negative vs high), p = 0.0128 (low vs med), p = 0.0021 (low vs high), p = 0.0067 (med vs high). Efnb3: p = 0.0075 (negative vs high) and 0.0164 (low vs high). Pcdh9: p = 0.0239 (negative vs low), 0.0021 (negative vs med), and 0.0074 (negative vs high). Slc24a2: p = 0.0427 (negative vs low), p = 0.0014 (negative vs med), p = 0.0099 (negative vs high), p = 0.0016 (low vs med), and p = 0.0309 (low vs high). Bar graphs show average ± SEM; *p < 0.05, **p < 0.001, ****p < 0.0001.

Article Snippet: Morphologic parameters, including average and maximum DAPI/MeCP2 intensity, ellipticity (oblate), and volume, were determined for each focus using the measurement tools in Imaris.

Techniques: Over Expression, Injection, Labeling, Control, Expressing, Comparison, Quantitative RT-PCR