Table 1.
List of antibodies used.
Figure 1.
In vitro differentiation of Mecp2 wild type ES cells is accompanied by large scale heterochromatin reorganization.
A. Shown are representative low and high (insets) magnification images of phase contrast (Ph) and DAPI DNA staining. Cells were kept in an undifferentiated state (day 0) in LIF containing medium. Differentiation of ES cells was induced by LIF withdrawal and plating into differentiation medium. Rosettes (arrow heads) indicated regions of neural differentiation, clearly visible as of day 7. Shortly thereafter EGFP positive neurons could be detected around and within rosettes and mark differentiating neural cells. At day 13 cells exhibit a tissue like growth. Bar: 10 µm. B. The neural identity of rosettes could be demonstrated by EGFP reporter expression driven by the neural tau promoter as of day 5 to 7 and most prominent after 13 days. Most EGFP positive cells are located within rosette structures, often surrounded by GFAP positive astroglia. Bar: 10 µm. C. Replicas of 100 DAPI stained nuclei were statistically analyzed and shown combined in a whisker box plot. Whiskers depict the 5–95 percentile of the confidence interval; the median is shown as a horizontal line within the box, and mean values are highlighted as white crosses. We noticed a highly significant (p<0.0001) decrease of mean chromocenter number per nucleus within the first two weeks of differentiation. Thereafter, the chromocenter number did not change significantly (p = 0.177). Statistical significance was tested with individual unpaired t-tests following Welch’s correction.
Figure 2.
MeCP2 becomes detectable in heterochromatic chromocenters at late differentiation stages.
A. Mecp2wt and Mecp2−/y tEG cells were differentiated, fixed, and immunostained with antibodies to MeCP2. Tau promoter driven EGFP expression highlights neuronal cells. Additional markers as beta tubulin III (neuronal cell) and GFAP (astroglia) allow discrimination of different cell populations. In immunofluorescence experiments MeCP2 protein is detected as of day 13 (left) and remains constant thereafter (middle) in EGFP+ cells. Line profiles across chromocenters highlight MeCP2 protein accumulation at these structures (black line) as well as their intense DAPI signal (blue line). Some GFAP+ astroglia revealed a relative weaker MeCP2 signal (black line) compared to neurons. Mecp2 deficient cells (right) were used as control. Bar: 10 µm. B. Immunoblot experiments demonstrate the increase of MeCP2 protein over time in wild type cells (wt) while Mecp2 deficient cells (−/y) show no signal. Beta actin was used as a control for equal loading.
Figure 3.
MeCP2 absence does not prevent neuronal and astroglia differentiation.
Confocal microscopy optical sections of Mecp2 wild type (left) and Mecp2 deficient cells (right) at differentiation day 13 revealed astroglia (GFAP+) and different neuronal subtypes (EGFP+, TH+, Sero+). Bar: 10 µm. A. Differentiated Mecp2 wild type and Mecp2 deficient cells exhibit GFAP positive astroglia (red) and EGFP positive neurons (green). Both labels marked mutually exclusive cell populations. B. The population of EGFP positive cells could be further subdivided into tyrosine hydroxylase (TH+) positive neurons (red). Those cells were found to be present in both Mecp2 wild type and Mecp2 deficient cells. While not all EGFP+ neurons (green) are TH+, all TH+ are positive for EGFP (yellow color in overlay). C. In addition to a TH+/EGFP+ subpopulation neurons, another serotonin positive (Sero+) subpopulation was detected in both cell lines. Similar to the situation described above, Sero+ cells (red) were always EGFP+ (green) while a considerable amount of EGFP+ cells were Sero−.
Figure 4.
Mecp2 deficient neurons and astroglia cells have significantly more chromocenters than wild type cells.
A. Representative confocal microscopy optical sections of differentiated Mecp2 wild type and Mecp2 deficient nuclei revealed characteristic differences in heterochromatin organization. Positive immunostaining for the astroglial marker GFAP (upper left) or for neuronal markers TH (lower left) and serotonin (lower right) indicated a differentiated cell state. In addition, the population of EGFP-only positive neurons was analyzed (upper right). Bar: 10 µm. B. Compared to wild type cells Mecp2−/y tEG nuclei (n = 100 each) revealed significantly more chromocenters, here visualized in whisker-box plots. Median values are shown as horizontal lines within the box, whiskers represent the 5–95 percentile, mean values are indicated as crosses, and outliers are visualized as dots. All analyzed samples show a highly significant (p<0.0001) difference of mean values. GFAP+/EGFP− wild type cells revealed an average chromocenter number of 5.8±2.4 compared to 10.2±2.3 chromocenters in Mecp2−/y tEG cells. The mean chromocenter number per nucleus in TH+/EGFP− Mecp2 wild type cells was 5.9±2.0 versus 10.4±2.3 chromocenters in Mecp2 deficient cells. In Sero+/EGFP+ wild type cells we found on average 4.9±1.4 chromocenter versus 9.3±2.0 chromocenters per nucleus in Mecp2 deficient cells. The EGFP+ only population, showing 5.4±2.0 chromocenters in Mecp2 wild type cells and 10.1±2.2 chromocenters in Mecp2−/y tEG cells, mirrored these tendencies. C. Unbiased analysis of data from three biological replicates (n = 100 per data set) for Mecp2 wild type (solid line) and Mecp2 deficient (dashed line) cells at different time points revealed significant decrease of chromocenters within the first two weeks of differentiation whereas extended differentiation time to 21 days did not result in further large scale heterochromatin remodeling. Error bars: 95% confidence intervals according to the mixed linear model.