Download X-Chromosome inactivation ratios affect wild-type

Human Molecular Genetics, 2004, Vol. 13, No. 12
DOI: 10.1093/hmg/ddh142
Advance Access published on April 28, 2004
1275–1286
X-Chromosome inactivation ratios affect
wild-type MeCP2 expression within mosaic
Rett syndrome and Mecp2 2/1 mouse brain
Daniel Braunschweig, Thomas Simcox, Rodney C. Samaco and Janine M. LaSalle*
Medical Microbiology and Immunology, Rowe Program in Human Genetics, School of Medicine, 1 Shields Avenue,
University of California, Davis, CA 95616, USA
Received March 14, 2004; Revised April 5, 2004; Accepted April 15, 2004
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in MECP2, encoding
methyl-CpG-binding protein 2 (MeCP2). The onset of symptoms in RTT is delayed until 6 – 18 months and 4 –6
months in the Mecp2 2/1 mouse model, corresponding to a dynamic and gradual accumulation of MeCP2
expression in individual neurons of the postnatal brain. Because of X chromosome inactivation (XCI), cells
within RTT females are mosaic for expression of the heterozygous MECP2 mutation. Using the targeted
Mecp2 mouse model, we investigated the effect of Mecp2 mutation on XCI and developmental MeCP2
expression in wild-type (wt)-expressing neurons by quantitative laser scanning cytometry. Mecp2 2/1
female mice exhibited uniform regional distribution of Mecp2 mutant-expressing cells in brain, but unbalanced XCI in the population, favoring expression of the Mecp2 wt allele. Interestingly, MeCP2 expression
in Mecp2 wt-expressing cells from Mecp2 2/1 mice was significantly lower than those from Mecp2 1/1 agematched controls. The negative effect of Mecp2 mutation on wt Mecp2 expression correlated with the percentage of Mecp2 mutant-expressing cells in the cortex. Similar results were observed in two RTT females with
identical MECP2 mutations but different XCI ratios. These results demonstrate that Mecp2-mutant neurons
affect the development of surrounding neurons in a non-cell-autonomous manner and suggest that environmental influences affect the level of MeCP2 expression in wt neurons. These results help in explaining the
role of XCI in the pathogenesis of RTT and have important implications in designing therapies for female
RTT patients.
INTRODUCTION
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder and a common genetic cause of severe mental retardation in females (1,2). Clinical features are not manifested
until around 6 –18 months of age and include the loss of vocalization skills and purposeful hand movements, deceleration of
head growth, ataxia, autistic features, seizures and breathing
abnormalities (3). RTT is caused by mutations in MECP2,
encoding methyl-CpG-binding protein 2 (MeCP2) (4).
Although MECP2 is ubiquitously expressed at the transcriptional level (5 – 7), tissue-specific and developmental regulation of MECP2 transcription and alternate polyadenylation
result in elevated MeCP2 expression limited to a subpopulation of neurons in the postnatal central nervous system
(CNS) (8 – 10). The MeCP2 binds to methylated DNA (11),
localizes to nuclear heterochromatin (12) and is predicted to
be a transcriptional repressor of methylated genes (13).
Several mouse models for RTT have been produced by
creating null or truncating mutations of Mecp2. Two groups
produced Mecp2-null mice by cre –lox recombinase technology that were targeted to either the whole mouse or restricted
to postmitotic neurons in the CNS (14,15). The onset of neurologic symptoms was delayed until 3– 6 weeks in the Mecp2 2/y
male mice and 4 –6 months in the Mecp2 2/þ female mice,
with lethality resulting by 10 weeks of age in the males. The
conditional neuronal Mecp2-null mice had the same phenotype, with a slightly delayed age of onset. A truncated
Mecp2 308/y mutant mouse also displayed many features of
RTT, including motor impairments, seizures, autistic behaviors and hypoactivity (16). The phenotype of the Mecp2 308/y
mice was less severe than that of the Mecp2-null mice and
*To whom correspondence should be addressed at: Medical Microbiology and Immunology, Rowe Program in Human Genetics, School of Medicine, 1
Shields Avenue, University of California, Davis, CA 95616, USA. Tel: þ1 5307547598; Fax: þ1 5307528692; Email: [email protected]
Human Molecular Genetics, Vol. 13, No. 12 # Oxford University Press 2004; all rights reserved
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was non-lethal. Furthermore, the Mecp2 3082/þ females displayed variability and delayed onset of symptoms compared
with the Mecp2-null models or RTT females. Overall these
mouse models appear to recapitulate the phenotype of RTT
in the mouse, although a major question that remains is why
the onset of symptoms in female Mecp2 2/þ mice is delayed
until adulthood in the mouse but occurs during infancy in
humans.
One hypothesis is that MeCP2 is required for neuronal
maintenance rather than maturation and therefore the onset
of symptoms may follow a chronological rather than developmental time line (14,15). Another possibility is that MeCP2
expression is developmentally regulated and not required
until postnatal CNS development during synaptogenesis
(8 – 10,17). In humans, MeCP2 protein expression shows
complex developmental and tissue-specific heterogeneity
(8 – 10), with a population of high-expressing (MeCP2hi)
neurons emerging in juveniles and continuing through adulthood in the cerebral cortex (10). Heterogeneity in the level
of MeCP2 within the CNS has also been observed in the
macaque (18), rat (19,20) and mouse (9,17). In mouse, developmental expression has been investigated previously by
immunoblot and immunohistochemistry, demonstrating a relatively stable expression during postnatal brain development
(9). Analysis of MeCP2 by immunoblot in isolated olfactory
bulb (17) or cerebellum (20) demonstrated increased
expression in adulthood, however, suggesting that changes
in MeCP2 expression may be evident within specific brain
regions. Although these previous studies have suggested a
role for MeCP2 in the maturational differentiation of CNS
neurons based on the timing of its expression, a quantitative
analysis of MeCP2 expression in different brain regions
during development has not been performed previously.
In addition to developmental regulation of MeCP2, X
chromosome inactivation (XCI) plays an important role in
the onset and severity of RTT symptoms (2). As MECP2/
Mecp2 is located on the X chromosome, female RTT patients
and Mecp2 2/þ mice are mosaic for expression of MECP2/
Mecp2 mutations. Although most RTT patients show
random XCI ratios in brain and blood (21 –23), some asymptomatic carriers of MECP2 mutations show extremely skewed
XCI (24). A recent report on XCI in the Mecp2 308/þ mouse
model demonstrated unbalanced XCI patterns favoring
expression of the wild-type (wt) allele that correlated with
phenotypic outcome (25). As MECP2 mutant cells are randomly distributed in the RTT brain (8), we hypothesized
that non-cell-autonomous mechanisms may influence the
level of MeCP2 expression in wt-expressing neurons.
In this study, we perform quantitative localization of
MeCP2 expression in sagittal mouse brain sections by laser
scanning cytometry (LSC). The LSC is a new technology
that not only quantifies immunofluorescence of contoured
cells within tissue sections, but also localizes each cell back
to the original tissue position (8). In this report, we use
LSC to demonstrate dynamic regional and developmental
expression and localization patterns of MeCP2 in brain that
were not fully evident by previous methodologies. In addition,
the combined use of LSC with tissue microarrays allowed us
to quantitate MeCP2 expression in subpopulations of cells
from multiple Mecp2 2/þ and control tissues on the same
slide. This quantitative approach allowed us to observe an
effect of Mecp2 mutation on expression of the wt allele in
Mecp2 2/þ mice that was not previously apparent. These
results suggest a model for the pathogenesis of RTT in
which the normal dynamic developmental expression and
function of MeCP2 is altered by MECP2 mutation by both
cell autonomous and non-autonomous mechanisms.
RESULTS
Quantitative localization of MeCP2 in the developing
mouse brain reveals dynamic and regional patterns of
expression
Because of previous evidence of complex regulation of
MeCP2 expression in different cellular subpopulations and
brain regions during mammalian brain development (8,9,17),
we decided to use LSC to quantitatively localize MeCP2
expression in whole mouse brain sections at 21 different
time points during embryonic and postnatal development.
The LSC analysis measures fluorescent intensity of individual
cells within tissue sections contoured on the basis of a nuclear
counterstain, as described previously (8). Figure 1 shows
representative analyses of eight different time points in
which consecutive sagittal sections taken 1 mm from the
midline were stained with hemotoxylin and eosin (H&E) to
identify brain regions within the x, y position scattergrams
(XY Pos) generated by LSC analysis that represent the slide
position of each contoured cell. Each slide was scanned
using identical non-saturating settings for the long red (Cy5
fluorescence) photomultiplier tube (PMT) so that quantitative
comparisons could be made between different time points.
Cell populations were gated and colored on the basis of
MeCP2 ‘max pixel’ histograms of whole brain. Max pixel is
the maximum fluorescent intensity of each contoured
nucleus, as described previously (8). Region 1 (blue) reflects
the low, but positive MeCP2 level observed in the late
embryonic time points. Regions 2 and 3 are the two dominant
populations found in the adult, reflecting the MeCP2lo (green)
and MeCP2hi (red) populations, respectively, as described previously (8). The colored populations are represented on the
LSC scattergram of x, y position and show regional differences
and changes in regional distribution throughout development.
In order to quantitate regional differences in MeCP2 fluorescence, six different identifiable brain regions (olfactory
bulb, cerebral cortex, hippocampus, cerebellum, medulla and
thalamus) were separately gated on the x, y scattergram (XY
Gated). Regional analysis demonstrated that high MeCP2 fluorescence in the neonatal mouse brain was limited to the medulla
and thalamus. By 1– 2 weeks of age, however, there was a dramatic decrease in MeCP2 fluorescence for both medulla and
thalamus that was primarily responsible for the overall decrease
in the whole brain from 1 to 2 weeks of age. From around 3 to
40 weeks of age, the MeCP2hi population (red) showed a
gradual reemergence in all regions of the brain.
A graphical representation of the developmental changes in
mean MeCP2 fluorescence of all contoured nuclei within
the whole brain for all 21 time points is shown in
Figure 2A. Each data point represents the mean+SEM from
four replicates of at least two mice for each time point. A
Human Molecular Genetics, 2004, Vol. 13, No. 12
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Figure 1. Dynamic regional expression of MeCP2 in the developing mouse brain revealed by laser scanning cytometry (LSC). Wild type (wt) C56BL/6 mouse brains were fixed at 21 different embryonic
and postnatal ages, sagittally sectioned, and analyzed for quantitative localization of MeCP2 immunofluorescence (Cy5 detection) of individual cells by LSC. Eight representative time points are shown to
demonstrate full LSC analysis, while mean MeCP2 levels for whole brain analyses of all samples and time points are graphed in Figure 2A. For each time point, the hemotoxylin and eosin (H&E) stained
parallel slide was used to determine the tissue localization of cells within the LSC scattergram of x,y position (XY Pos). For embryonic and early postnatal time points, the whole head was sectioned and the
brain region gated on the x,y position scattergram; black regions represent non-brain cells that were excluded from analysis. Histograms representing MeCP2 fluorescence (x-axis) and cell count (y-axis) for
the whole mouse brain section (Whole Brain) are shown for LSC analyses with equivalent long red PMT detection for all time points to quantitate developmental differences in MeCP2 expression. Histograms are gated and colored into five regions based on MeCP2 fluorescence: region 1 (blue) represents the late embryonic expression of MeCP2, set at the right half max of the E16 time point: region 2
(green) represents the previously described MeCP2lo population, set at the right half max of the adult P40 week time point; region 3 (red) represents the previously described MeCP2hi population, and
includes the remainder of the cells. In order to examine regional differences in these populations, different anatomical structures identified from H&E staining were gated on the x,y position scattergram (XY
Gated). Histograms of MeCP2 fluorescence (x-axis) and cell count (y-axis) for each separately gated region are shown for each time point. The total of number of cells contoured are shown for each
histogram. Dynamic changes were observed in MeCP2 expression-based populations in different brain regions throughout development. A large population of MeCP2hi cells was observed in the perinatal
medulla and thalamus that decreased by 1 week of age and gradually re-emerged from 4 to 40 weeks of age in all brain regions.
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Human Molecular Genetics, 2004, Vol. 13, No. 12
3-fold increase in MeCP2 max pixel mean was observed from
the earliest (E10 or 29d) to the latest (E19 or 0d) embryonic
time point. This was followed by a rapid equivalent decrease
in early postnatal life, then a rapid 3-fold increase from 5d
to 21 days. From around 3 to 40 weeks (21 – 280 days) of postnatal life, there was a gradual 2-fold increase in MeCP2 max
pixel mean, reflecting the accumulation of the MeCP2hi population in all regions of the brain, as demonstrated in Figure 1.
As the max pixel measurement for quantitating MeCP2
immunofluorescence used in Figures 1 and 2A could reflect
changes in localization of MeCP2 within the nucleus as well
as the level of MeCP2 expression, we sought to determine
MeCP2 expression and nuclear localization by additional
LSC analyses and immunoblot. The LSC analysis of long
red integral is the sum of all the pixel intensities inside
the nuclear contour (similar to the ‘area under the curve’
measurement commonly used in image quantitation) and
reflects the total amount of MeCP2. To normalize for cell
size, the MeCP2 ‘integral’ was divided by the nuclear area
for each contoured cell and the results from the whole brain
analyses are plotted in Figure 2B (black circles and solid
line). A .4-fold spike was still observed in the neonatal
brain for the MeCP2 integral over nuclear area measurement,
but unlike the MeCP2 max pixel measurement in Figure 2A,
no increase was observed from 1 to 3 weeks of age. These
results suggested that the second increase in MeCP2 max
pixel mean observed from 5 to 21 days in Figure 2A was
due to MeCP2 localization changes and not due to an increase
in the overall expression. Interestingly, there was a corresponding increase in the nuclear area (open squares and
dotted line) during the first 2 weeks of postnatal age,
suggesting that global nuclear organization changes occurring
during this time frame may reflect the difference in the two
analyses.
Immunoblot analysis of MeCP2 expression using protein
extracted from whole mouse brains at 17 different time
points was performed as an independent method (Fig. 2C).
Following detection with anti-MeCP2, blots were re-probed
with anti-GAPDH for semi-quantitative analysis of MeCP2
expression. The quantitative results from four different immunoblots are shown in Figure 2D, demonstrating a similarly
rapid 5-fold increase in MeCP2 expression in the perinatal
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brain as was observed for the analysis of MeCP2 integral normalized to nuclear area in Figure 2B (closed circles and solid
line). In contrast, the gradual 2-fold increase in MeCP2
expression observed by LSC analysis from 3 to 40 weeks
(Fig. 2A and B) was not observed by immunoblot analysis
(Fig. 2D). The LSC’s ability to quantitate expression levels
of individual cells unlike the whole protein analysis by immunoblot may explain the discrepancy. These combined results
demonstrate that the dynamic changes observed in MeCP2
fluorescence observed by LSC reflect changes in both the
level of protein expression and the changes in nuclear localization of heterochromatin.
Mecp2 2/1 mice show uniform distribution of MeCP2negative cells in brain
Because of the important influence XCI ratios have on the
development of RTT in humans (22), we were interested in
quantitating and localizing the Mecp2 mutant-expressing
cells within females heterozygous for the Mecp2-null
mutation. Two different Mecp2-null strains were used for
these studies. The Mecp2 tm1.1Bird strain has excised Mecp2
exons 3 and 4 (15), while the Mecp2 tm1.1Jaestrain has only
exon 3 deleted (14). Male Mecp2 2/y mice were used as a
negative control for immunofluorescence as they have been
determined previously to have an absence of MeCP2
expression (14,15). Consistent with this expectation,
Mecp2 tm1.1Bird/y mice had a level of MeCP2 immunofluorescence equivalent to the immunoglobulin G (IgG) control
(Fig. 3A) and were used to set the region for determining
the MeCP2-negative population. For whole brain regional
analysis of MeCP2-negative cells in heterozygous females,
sagittal brain sections from two Mecp2 tm1.1Bird/þ mice were
analyzed for regional MeCP2 expression as in Figure 1. A
graph of the combined results of the MeCP2-negative cells
for different brain regions is shown in Figure 3B. Although
there existed some variation in the percentage of MeCP2negative cells in differently gated brain regions for each individual, no region was significantly different than the whole
brain by t-test. These results demonstrate uniform regional distribution of MeCP2-negative cells in the mouse brain.
Figure 2. Quantitation of MeCP2 expression versus nuclear localization changes in whole mouse brain by LSC and immunoblot analyses. (A) Quantitation of
developmental changes in MeCP2 fluorescent intensity by LSC max pixel analysis of whole sagittal brain sections for 20 different time points. Long red max
pixel represents the highest intensity pixel within the contoured nuclei and is influenced by localization of MeCP2. The mean long red max pixel from the histograms in Figure 1 (Whole Brain) are plotted on the y-axis, with chronological age in days on the x-axis. The results are graphed as the mean + SEM of four
replicate experiments from at least two mice per time point. A sharp rise in the MeCP2 max pixel mean was observed in the late embryonic to early postnatal
time points (21 to 1d), followed by a decline in the first week of life, a second rise from 1 week to 3 weeks, then a gradual rise from 4 to 40 weeks. (B) The LSC
quantitation of total levels of MeCP2 per nucleus compared with nuclear area. The LSC analysis of MeCP2 integral values divided by nuclear area for each
contoured nucleus is plotted as filled circles, representing the mean + SEM and solid trendline. Long red integral is the addition of all pixel intensities
within the nuclear contour, normalized for cell size by dividing by the nuclear area. The first peak of MeCP2 expression in perinatal development is the
most pronounced change, while the second increase in MeCP2 max pixel values observed from 1 to 3 weeks in A was not observed. Nuclear area measurements
are also plotted (open squares and dotted trendline) and demonstrate a major increase from P1 to P10, with relatively stable values beyond this time frame. The
coincidence of nuclear area increase and MeCP2 max pixel values suggests that changes in MeCP2 localization to large nuclear heterochromatin sites occurs
during this time frame. However, a gradual 2-fold increase in MeCP2 integral normalized to nuclear area was also observed from P10 to P280, consistent with the
emergence of the MeCP2hi population, suggesting that changes in expression and localization of MeCP2 occur during mouse adulthood. (C) Immunoblot analysis
of whole mouse brain MeCP2 expression levels was performed for 17 different time points. MeCP2 was observed as a single 76 kDa band in all lanes but relative
expression varied with development. The blot was re-probed with an antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control, which ran as
a single 37 kDa band. (D) Band intensities from three replicate immunoblots were quantitated and plotted as MeCP2/GAPDH (y-axis) versus chronologic age (xaxis). The solid trendline appears similar to the solid line in B representing the LSC total nuclear MeCP2 measurements except the gradual 2-fold increase was
not observed from P10 to P280. Thus, the LSC may be more sensitive to detecting MeCP2 expression differences because of the sampling of individual cells.
Trendlines were plotted based on a moving average of two points on each timecourse.
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Figure 3. Precise determination of MeCP2-negative populations in two Mecp2-null mouse models and regional distribution in whole brain. (A) The LSC analysis
of cerebral cortical samples containing the different genotypes of two different RTT mouse models was performed at both saturating and non-saturating settings.
Saturating conditions were used to compare Mecp2 2/y samples to the IgG negative control (black overlay) so that the region for determining the MeCP2-negative population (region 1, gray) could be set (top). The middle panel shows representative histograms from female Mecp2 2/þ mice in which the percentage of
MeCP2-negative population was determined using region 1 and saturating conditions so that the two expression peaks were noticeably separate. The bottom
panel shows the same female samples under non-saturating settings. Region 1 was set to include the percentage of MeCP2-negative population previously determined for each tissue. Cells in region 2 (MeCP2-positive) were separately gated to obtain the values for MeCP2 and histone H1 expression in Figure 5. (B) Brain
regional localization of MeCP2-negative populations was determined from whole brain sagittal sections of two Mecp2 tm1.1Bird/þ mice with 30% MeCP2-negative
cells. Regional gating was performed as in Figure 1, and the results shown are the mean + SEM of the two samples for each region. No significant differences
were observed in the distribution of MeCP2-negative cells in any region compared with the whole brain.
The percentage of MeCP2-negative calculated from
each Mecp2 2/1 brain sample inversely correlates with
MeCP2 expression in wt-expressing cells
If Mecp2/MECP2 mutations acted by a completely cellautonomous mechanism in mosaic females, only cells expressing the mutant allele would exhibit a phenotype. In light of
the complex regulation of MeCP2 expression in postnatal
development, we hypothesized that MeCP2 expression levels
and/or localization may be affected in the MeCP2-positive
expressing cells of females heterozygous for MECP2/Mecp2
mutation. To test the hypothesis in mouse, a tissue microarray
was prepared containing triplicate cores of cerebral cortex
from five Mecp2 tm1.1Bird/þ, five Mecp2 tm1.1Jae/þ, and three
Mecp2 þ/þ controls (P20 –27w); one Mecp2 tm1.1Bird/y, and
one Mecp2 þ/y littermate (P10w); one Mecp2 tm1.1Jae/y and
one Mecp2 þ/y littermate control (P10w). Serial sections
derived from tissue microarrays were analyzed for C-terminal
anti-MeCP2 (four replicates), rabbit IgG (negative control, one
replicate) or anti-histone H1 (positive control, one replicate)
immunofluorescence by LSC. Tissue microarrays minimize
inter-slide variability and standardize tissue sampling by combining multiple tissues on the same slide (26).
Figure 3A shows how the percentage of MeCP2-negative cells
was determined for each tissue sample on the microarray based on
the appropriate Mecp2 2/y negative control. As the large dynamic
range of MeCP2 expression limited the resolution of MeCP2negative cells from the MeCP2lo population, saturating LSC
settings were used to precisely determine the percentage of
MeCP2-negative cells (Fig. 3A, top four histograms). Two distinct peaks could be observed only in Mecp2 2/þ samples, representing MeCP2-negative (Mecp2 mutant-expressing) and
MeCP2-positive (Mecp2 wt-expressing) cells. The gate for
MeCP2-negative cells (region 1, colored in gray) was set to the
right half maximum of the Mecp2 tm1.1Bird/y histogram (top left)
and was used to determine the percentage of MeCP2-negative
cells in each of the Mecp2 tm1.1Bird/þ tissues (as shown in the
middle left histogram) in four replicate experiments
(mean + SEM graphed in Fig. 4A and B, y-axes). The mean percentage of MeCP2-negative cells ranged from 20 to 39% for the
five Mecp2 tm1.1Bird/þ mice, somewhat lower than the overall
expected 50%.
In contrast to the Mecp2 tm1.1Bird/y tissues that were negative
for MeCP2 immunofluorescence, the Mecp2 tm1.1Jae//y mice
had a background immunoreactivity (Fig. 3A, top right
panel, filled histogram) that was higher than the IgG control
Human Molecular Genetics, 2004, Vol. 13, No. 12
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Figure 4. XCI ratios influence the MeCP2 expression in Mecp2 wt-expressing cells of mosaic female mice. (A and B) Scattergrams plotting the percentage of
MeCP2-negative population (mean + SEM of four replicates) on the y-axis versus the MeCP2 max pixel (A) or mean intergral/nuclear area (B) values
(mean + SEM of four replicates) of the gated MeCP2-positive population for each mouse sample on the tissue microarray. Mecp2 þ/þ mice (open squares)
were analyzed identically to the 10 Mecp2 2/þ mouse samples (closed circles) and show higher MeCP2 expression by both measurements. An inverse correlation
was observed between the percentage of MeCP2-negative cells and MeCP2 expression in the wt-expressing cells of Mecp2 2/þ mice (P , 0.05 by F-test for both
A and B). (C) Fluorescent micrographs of MeCP2 immunofluorescence showing absence of staining in nuclei from Mecp2tm1.1Bird/y cerebral tissue, reduced or
absent expression in Mecp2tm1.1Bird/þ cerebral nuclei, and normal MeCP2 expression in Mecp2 þ/þ control tissue. Images were captured using identical exposure
and deconvolution settings. Examples of the MeCP2-negative population (closed arrows), MeCP2lo nuclei (open arrows), and MeCP2hi nuclei (diamond arrows)
are shown. Although the Mecp2tm1.1Bird/þ brain tissue shows a majority of MeCP2-positive (wt-expressing) nuclei, they are primarily of the MeCP2lo phenotype.
(overlay). A similar background immunoreactivity can be
explained as a truncated mutant protein by immunoblot for
the Mecp2 tm1.1Jae/y but not for the Mecp2 tm1.1Bird/y mice
(data not shown) and has been described previously (27).
The gate for region 1 (gray, MeCP2-negative cells) was therefore set at the right half max of the saturated Mecp2 tm1.1Jae/y
control (top right histogram), which also separated the
lowest peak in the saturated Mecp2 tm1.1Jae/þ samples
(middle right panel) and was used to determine the percentage
of MeCP2-negative cells in the four replicate experiments
(mean + SEM graphed in Fig. 4A and B, y-axes). The mean
percentage of MeCP2-negative cells ranged from 18 to 37%
for the five Mecp2 tm1.1Jae/þ mice. The overall mean for the
10 Mecp2 2/þ mice on the tissue microarray was 29.9 + 7.5%,
which was significantly lower than the expected 50% by
t-test (P , 0.0001). These results demonstrate unbalanced
XCI patterns in favor of inactivating the mutant allele.
The mean percentage of MeCP2-negative calculation
obtained for each tissue was then used to set the gates for
the histograms of MeCP2 fluorescence obtained at non-saturating LSC settings to obtain quantitative measurements of
MeCP2 in the Mecp2 wt-expressing cells (MeCP2-positive)
of each tissue, as shown in the bottom panel of Figure 3A
(MeCP2-negative colored in gray, MeCP2-positive colored
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in black). The values of MeCP2 max pixel (Fig. 4A) and
MeCP2 integral/nuclear area (Fig. 4B) were obtained for the
gated MeCP2-positive population for each tissue (x-axes,
mean + SEM from four replicates). For both mouse models,
the Mecp2 wt-expressing cells (MeCP2-positive) from the
Mecp2 2/þ tissues (filled circles) showed lower MeCP2
expression than the equivalently gated MeCP2-positive cells
from Mecp2 þ/þ tissues (open squares). The percentage of
MeCP2-negative cells in the Mecp2 2/þ tissues inversely
correlated with both the MeCP2 max pixel (P , 0.05 by
F-test) and the MeCP2 integral/nuclear area (P , 0.05, by
F-test) of the MeCP2-positive cells in the same tissue
samples. In contrast, the analysis of histone H1 values in the
MeCP2-positive cells showed no significant correlation with
the percentage of MeCP2-negative cells by F-test (data not
shown). Representative images of MeCP2 immunofluorescence are shown in Figure 4C, demonstrating the low
MeCP2 expression characteristic of the MeCP2-positive
nuclei from Mecp2 2/þ brain tissue compared with Mecp2 þ/þ
controls. These results demonstrate that XCI ratios in
Mecp2 2/þ mice can influence the wt Mecp2 expression.
Human RTT brain samples also show an
effect of XCI on wt MECP2 expression
To investigate whether a similar effect of mosaic MECP2
mutation on MeCP2 expression in wt-expressing neurons
could be observed in human female RTT brain samples, we
created a human post-mortem brain tissue microarray containing six replicate cores sampled from frontal cerebral cortex of
three patients with known MECP2 mutations and triplicate
samples of control subjects of various ages. RT 1238 was
from a previously described male subject with a
MECP2 1154(del32) mutation that died at 21 months of age
(28). The MeCP2 protein from this patient was predicted to
lack 100 amino acids at the C terminus, including the C-terminal antibody epitope, and therefore this hemizygous male was
used as the control for determination of the MeCP2-negative
population. Figure 5A demonstrates that when the tissue
microarray stained with anti-MeCP2 was scanned using saturating LSC settings for long red max pixel, the RT 1238 gated
histogram (filled, top) overlapped with the non-specific antibody control (black overlay, top). Region 1 (gray, MeCP2negative population) was set to the right half maximum of
the RT 1238 histogram and was used to determine the percentage of MeCP2-negative cells in two RTT female samples
with MECP2 R168X truncating mutations at the same saturated
settings (middle histograms). RT 3393 and RT 4312 showed
67 and 30% MeCP2-negative cells, respectively. These percentages were used to set region 1 under unsaturated LSC
setting (lower histograms), so that the MeCP2-positive cells
(region 2, black) could be analyzed for MeCP2 max pixel
and intergral/nuclear area values. The same analysis was performed on samples from a group of six controls ranging from
11 to 30 years of age. Figure 5B demonstrates that while
MeCP2-positive cells from both RT 3393 and RT 4312 had
significantly lower MeCP2 max pixel values than controls,
the difference was much more significant in RT 3393
than in RT 4312 (P , 0.001 and P , 0.05, respectively).
For the MeCP2 integral/nuclear area measurement (Fig. 5C),
MeCP2-positive cells from RT 3393, but not from RT 4312,
were significantly different from controls (P , 0.01). In
contrast, similar measurements of histone H1 were not significantly different between MeCP2-positive cells of patients and
controls (Fig. 5B and C). These results from human RTT
samples are therefore in agreement with the findings from
Mecp2 2/þ mice in showing a non-cell-autonomous effect of
MECP2/Mecp2 mutation on wt MECP2 expression that correlates with the percentage of MeCP2 mutant-expressing cells.
Interestingly, the phenotype of RT 3393 (diagnosis at
4 months, death at 12 years) appeared to be more severe
than that of RT 4312 (diagnosis at 18 months, death at 24
years). These results suggest that XCI can influence the RTT
phenotype by both cell-autonomous and non-autonomous
mechanisms.
DISCUSSION
In this report, we have used the quantitative method of detecting MeCP2 immunofluorescence by LSC in order to understand the normal developmental and regional expression of
MeCP2 and to separately analyze MeCP2 mutant- and wt.expressing cells in human RTT samples and the Mecp2 2/þ
mouse model of RTT. These analyses have allowed several
novel observations to be made relevant for understanding
the pathogenesis of RTT. First, we demonstrate that MeCP2
expression and localization is dynamically controlled at
several stages during mammalian brain development. A timeline is shown in Figure 6A to summarize the developmental
changes in MeCP2 and the phenotypic manifestations in the
RTT mouse model. Although MeCP2 is expressed in virtually
all murine cells and developmental stages, the level rises
sharply in the medulla and thalamus around birth, then falls
sharply by the first postnatal week. Between 1 and 3 weeks
of age, MeCP2 immunofluorescence intensity rises sharply
due to increased localization to multiple heterochromatic
nuclear foci, characteristic of the MeCP2lo neurons. The phenotype of the Mecp2 2/y mice becomes apparent soon after,
from around 3 to 6 weeks of age (14,15), suggesting that
MeCP2 has an essential function at this developmental
stage. The next change occurs in MeCP2 expression level in
individual neurons throughout all brain regions, resulting in
the gradual accumulation of the MeCP2hi population. The
delay of a noticeable phenotype in the Mecp2 2/þ mice until
adulthood (4 – 6 months) could be due to this continued
increase in the MeCP2hi population throughout adulthood
and the specific requirement for MeCP2 in this population.
Although the developmental expression of MeCP2 has been
described previously in several mammals (9,17 –20), our study
of wt mice more completely investigates the changes at the
single-cell level throughout the brain and at 20 different
ages and identifies more dynamic changes in MeCP2 than
were realized previously. Prior investigations performed by
immunohistochemistry have identified more intensely stained
neuronal nuclei at later postnatal ages, but these were not
quantitated previously (9,18,20). Prior immunoblot analyses
of olfactory bulb (17), cerebellum (20) and hippocampus
(29), but not whole brain extracts (9), identified increased
MeCP2 expression with postnatal age, consistent with our
Human Molecular Genetics, 2004, Vol. 13, No. 12
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Figure 5. Analysis of RTT females with MECP2 mutations for evidence of an effect of XCI on wt MECP2 expression. (A) The LSC analysis of cerebral samples
on a human tissue array containing samples from one male and two females with MECP2 truncation mutations analyzed at saturating (top and middle panels) and
non-saturating (bottom) conditions. A two-step analysis was performed as for the mouse tissues in Figure 3A, to determine the percentage MeCP2-negative for
each female sample, and then determine the MeCP2 and histone H1 expression of the MeCP2-positive cells in the two RTT female samples. (B) MeCP2 mean
max pixel values of the MeCP2-positive cells were significantly lower compared with those of age-matched controls, although the significance was higher for
sample RT 3393 with 67% MeCP2-negative cells compared with RT 4312 with only 30%. (C) MeCP2 mean integral/nuclear area measurements were significantly lower than controls for the MeCP2-positive population of the RT 3393 sample, but not the RT 4312 sample. P , 0.05; P , 0.01; P , 0.001.
results showing an increased MeCP2hi population in these
regions (Fig. 1). Our immunoblot analyses of MeCP2 in
whole brain extracts (Fig. 2C and D) also failed to show
MeCP2 expression increase in mouse adulthood, presumably
because the individual subpopulations or brain regions were
not fractionated. Another possibility could be that immunofluorescence-based assays detect changes in epitope exposure
rather than expression changes. We believe this to be an unlikely explanation because MECP2 transcripts are also significantly increased in the MeCP2hi population (30) and
immunoblots of specific brain regions also demonstrate postnatal increases in MeCP2 reactivity (17,20,29). In addition,
our protocol involves boiling tissue sections for 1 h in
antigen retrieval solution. Changes to the pH and citrate concentrations of the antigen retrieval solution do not significantly
alter MeCP2 immunofluorescence (data not shown),
suggesting that protein – protein interactions are unlikely to
affect antibody binding in our assay. Two recent reports
have described a second isoform of MeCP2 arising from
alternative splicing of exon 2 and resulting in a change in
the N terminus that is indistinguishable from the previously
described isoform by immunoblot (31,32). As our study utilized a C-terminal reactive antibody for MeCP2, both
MeCP2 isoforms were detected most likely by immunofluorescence and immunoblot. Future studies using isoform-specific
reagents would be useful in determining if the dynamic
MeCP2 expression changes are unique to the MeCP2B
(MeCP2a in mouse) isoform predominant in brain (31,32).
By examining MeCP2 expression levels in the wt-expressing cells of Mecp2 2/þ mosaic females we have also made
several significant observations regarding the role of XCI on
the pathogenesis of RTT. We demonstrate that the range of
MeCP2 mutant-expressing cells from adult Mecp2 2/þ mice
from two different mouse strains was 18 – 39% (mean
29.9%), significantly lower than the expected 50%. These
results demonstrate an unbalanced XCI patterns in adult
Mecp2 2/þ mice, favoring expression of the wt allele. Our
results confirm those of a recent study examining XCI in the
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Human Molecular Genetics, 2004, Vol. 13, No. 12
Figure 6. Models for effect of developmental dynamics of MeCP2 expression
and localization and the role of XCI on the pathogenesis of RTT. (A) Developmental timeline of MeCP2 expression and localization changes in the wt
mouse and the development of the RTT-like phenotype in the Mecp2-null
mouse model. (B) Model for the role of a non-cell-autonomous mechanism
involved in the maturation of neurons from the MeCP2lo to MeCP2hi phenotype. In Mecp2 þ/þ mice, immature MeCP2lo neurons receive signals from surrounding neurons (arrows) that trigger them to become mature MeCP2hi
neurons. In mosaic Mecp2 2/þ and RTT females, however, the Mecp2/
MECP2 mutant cells surrounding the wt-expressing neurons limit the maturational signals, so fewer wt neurons mature to the MeCP2hi phenotype. The
higher the percentage of Mecp2 mutant-expressing cells in the brain, the
greater the influence of this ‘bad neighborhood’ effect.
Mecp2 308/þ mouse model (25). The selection against Mecp2
mutant-expressing cells could occur at the level of XCI in
the early embryo, but a more likely explanation is a positive
selection of the wt-expressing cells compared with themutant-expressing cells, similar to that observed in lymphocyte cultures from RTT blood (33). As many questions
remain with regard to the timing and occurrence of the XCI
bias in Mecp2 2/þ mice, future experiments examining XCI
ratios in different mouse strains, tissues and developmental
stages are warranted.
Our most interesting and novel observation from these
studies is that Mecp2 mutant-expressing cells influence the phenotype of the wt-expressing cells in Mecp2 2/þ female mice by
a non-cell-autonomous mechanism. The data presented in
Figure 4 demonstrate that the percentage of MeCP2-negative
cells inversely correlates with MeCP2 expression in the
MeCP2-positive cells. These data demonstrate that cell extrinsic factors influence the complex developmental control of
MeCP2 expression during postnatal brain development. A
model for this hypothesis is shown in Figure 6B. In wt
Mecp2 þ/þ mice, immature neurons of the MeCP2lo phenotype
would receive synaptic stimuli from surrounding neurons that
cause them to undergo a maturational differentiation to
become MeCP2hi neurons. In the Mecp2 2/þ brain, however,
the wt-expressing neurons are in a ‘bad neighborhood’ being
surrounded by neurons lacking functional MeCP2. They
would therefore receive fewer extrinsic stimuli, resulting in
fewer neurons progressing to the MeCP2hi phenotype and
resulting in lower overall MeCP2 expression. Brains containing
fewer MeCP2 mutant-expressing neurons would therefore be
the least affected. Our recent evidence for multiple pathways
affecting the level of MeCP2 expression in several neurodevelopmental disorders is also supportive of this model (30).
Two recent studies demonstrating brain-derived neurotrophic factor (BDNF) as a potential neuron-specific target
for MeCP2 may be relevant to our model, as BDNF is a
soluble factor that promotes survival and maturation of
neurons (27,34). Cortical neurons from Mecp2-null mice had
higher levels of BDNF than wt control neurons prior to stimulation, but not following KCl-mediated activation, suggesting
that MeCP2 is involved in the repression of BDNF prior to
neuronal activation (27). Although increased levels of BDNF
may be expected to promote rather than prevent neuronal
maturation, the direct role of BDNF and other potential gene
targets of MeCP2 in the pathogenesis of RTT remain to be
investigated.
Two brain samples from female RTT patients with MECP2
mutations also showed evidence for a non-cell-autonomous
mechanism influencing MeCP2 expression in wt-expressing
cells. The most striking evidence was from RT 3393,
showing 67% MECP2 mutant-expressing cells, higher than
that observed in any of the Mecp2 2/þ mice. The discordance
between random XCI patterns generally found in RTT patient
brain and blood compared with the non-random patterns
observed in Mecp2 2/þ mice could be due to a sampling
bias in the human population towards those that meet the diagnostic criteria for RTT (25). Perhaps MECP2 mutations could
be found in additional females with milder forms of mental
retardation and favorable XCI ratios. Finally, our data may
have implications for the potential treatment of RTT patients
if the cell extrinsic factors regulating the level of MeCP2
expression in wt-expressing neurons could be characterized
and mimicked pharmacologically.
MATERIALS AND METHODS
Mouse tissue
Mecp2 tm1.1Bird/þ (Jackson Laboratories) and Mecp2 tm1.1Jae/þ
(MMRRC) were mated with wt C57BL/6J mice (Jackson Laboratories) to produce the four possible genotypes. Genotyping
was performed using protocols provided by the commercial
vendors. An additional multiplex PCR was performed with
genomic DNA to confirm the presence or absence of Mecp2
exon 4 in Mecp2 tm1.1Bird/þ mice. PCR conditions were as
follows: 0.02 mM of primer pair 105 (50 -GTGAAGGAGTC
TTCCATACGGTC-30 ) and 106 (50 -TCTCCTTGCTTTTAC
GCCC-30 , 183 bp product) and 0.4 mM of primer pair 107
Human Molecular Genetics, 2004, Vol. 13, No. 12
(50 -ATGGCCTGCCAGAGTCGT-30 ) and 108 (50 -CAATTGGAACTGTCAACTACGGTT-30 , 618 bp product) with PCR
amplification performed through 33 cycles of 35 s at 958C,
45 s at 59.28C and 1 min at 728C. Mice were euthanized at
different developmental time points (P1 corresponds to day
of birth). Whole brain specimens were cut sagittaly at the
midline using a sterile scalpel and one half was placed in
10% neutral buffered formalin for 2 –3 days and then
embedded in paraffin blocks; the other half was placed in
Trizol and stored at 2808C for protein and nucleic acid
extractions. Sections of 1 mm were removed from each
block followed by sectioning at 5 mm for experimental
slides. The H&E slides were prepared for each brain specimen
to aid in identification of brain regions. Epitope exposure was
carried out as described previously (8).
Tissue microarray construction
Triplicate 600 mm cores of cerebral cortex were removed from
five Mecp2 tm1.1Bird/þ, five Mecp2 tm1.1Jae/þ and three Mecp2 þ/þ
controls (P20 –27w); one Mecp2 tm1.1Bird/y,and one Mecp2 þ/y
littermate (P10w); one Mecp2 tm1.1Jae/y and one Mecp2 þ/y
littermate control (P10w) paraffin ‘donor’ blocks based on
H&E slide reference and inserted into a recipient paraffin
block using a manual tissue arrayer (Beecher Instruments).
Resultant blocks were incubated at 508C for 10 min to set the
cores and serial 5 mm sections were cut from the array blocks
and stained as above for immunofluorescence. An H&E slide
was made to verify tissue sampling. A human developmental
tissue microarray described previously (10), was supplemented
with six cerebral cortex samples from three RTT patients and
processed as above. Use of human samples was approved by
the institutional IRB (#200210814-1).
Immunofluorescence
Anti-MeCP2 C terminus (Affinity Bioreagents), mouse IgG,
anti-histone H1 (Upstate Biotechnology) were diluted 1 : 100
in IF stain buffer [phosphate buffered saline (PBS) 1% fetal
calf serum/0.5% Tween-20] and incubated on slides for 2 h
at 378C, followed by three washes in PBS/0.5% Tween-20.
Cy5-labeled secondary antibody to rabbit IgG (Jackson Immunoresearch) or Cascade Blue-labeled secondary antibody to
mouse IgG (Molecular Probes) were diluted 1 : 100 in IF
buffer containing 10 mg/ml Sytox Green (Molecular Probes)
and incubated on slides for 1 h at 378C, followed by three
washes in PBS/0.5% Tween-20. Slides were mounted in
50% glycerol with 10 mg/ml Sytox Green and sealed with
nail polish.
LSC and fluorescence microscopy
Slides stained with anti-MeCP2, anti-histone H1 (positive
control) or IgG negative control were scanned on an LSC-1
under the 20 objective using appropriate settings (CompuCyte,
Cambridge, MA, USA). Cells were contoured on the basis of
Sytox Green nuclear fluorescence, optimized for each experimental and control slide pair to include as many individually
contoured cells as possible. Cell clusters were gated out of
analysis as described previously (8). Slides were visualized
1285
using a 60 oil immersion lens on a Zeiss Axioplan II microscope and images collected using digital deconvolution software as described previously (8).
Immunoblotting
Protein was isolated from mouse brain using TRIzol (Invitrogen) according to manufacturers protocol. An amount of 5 mg
of protein was loaded into each lane in pre-cast 4– 15% Tris –
HCl polyacrylamide gels (Biorad), and 10 ml of Kaleidoscope
pre-stained standards (Biorad) were loaded as a molecular
weight marker. Gels were electrophoresed and transferred to
nitrocellulose membranes. Membranes were washed with
tris-buffered saline (TBS) (Biorad) and blocked with 5%
non-fat dry milk (Carnation) in TBS. Primary antibody incubation with anti-MeCP2 (Affinity) diluted 1: 2000 in TBS
was done overnight at 48C. Blots were then washed in TBS
and incubated with a 1 : 2500 dilution of goat anti-rabbit
horseradish peroxidase (HRP) (Biorad) for 40 min at room
temperature. After washing in TBS, Supersignal West Pico
chemiluminescent substrate (Pierce) was added, and the
results recorded on photographic film. Glyceraldehyde 3-phosphate dehydrogenase (Advanced Immunochemical) was
diluted 1 : 600, then detected with goat anti-mouse HRP
(Biorad) in a 1 : 10,000 dilution, as above.
ACKNOWLEDGEMENTS
The authors thank M. Lalande for critical review of the manuscript and W.P. Robinson for statistical advice. This work was
supported in part by the NIH (R01HD/NS41462), the Rett
Syndrome Research Foundation, and the U.C. Davis
M.I.N.D. Institute. Mecp2 tm1.1Jae mice were obtained from
the Mouse Mutant Regional Resource Center at U.C. Davis
(supported in part by NIH U42 RR14905). Human tissue
samples were obtained from the University of Maryland
Brain and Tissue Bank for Developmental Disorders (supported by NIH N01-HD-1-3138) and the Harvard Brain
Tissue Resource Center (supported in part by PHS MH/NS
31862).
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