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PLoS One
2012 Jan 01;73:e33153. doi: 10.1371/journal.pone.0033153.
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Cell-autonomous alterations in dendritic arbor morphology and connectivity induced by overexpression of MeCP2 in Xenopus central neurons in vivo.
Marshak S
,
Meynard MM
,
De Vries YA
,
Kidane AH
,
Cohen-Cory S
.
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Methyl CpG binding protein-2 (MeCP2) is an essential epigenetic regulator in human brain development. Mutations in the MeCP2 gene have been linked to Rett syndrome, a severe X-linked progressive neurodevelopmental disorder, and one of the most common causes of mental retardation in females. MeCP2 duplication and triplication have also been found to affect brain development, indicating that both loss of function and gain in MeCP2 dosage lead to similar neurological phenotypes. Here, we used the Xenopus laevis visual system as an in vivo model to examine the consequence of increased MeCP2 expression during the morphological maturation of individual central neurons in an otherwise intact brain. Single-cell overexpression of wild-type human MeCP2 was combined with time-lapse confocal microscopy imaging to study dynamic mechanisms by which MeCP2 influences tectal neuron dendritic arborization. Analysis of neurons co-expressing DsRed2 demonstrates that MeCP2 overexpression specifically interfered with dendritic elaboration, decreasing the rates of branch addition and elimination over a 48 hour observation period. Moreover, dynamic analysis of neurons co-expressing wt-hMeCP2 and PSD95-GFP revealed that even though neurons expressing wt-hMeCP2 possessed significantly fewer dendrites and simpler morphologies than control neurons at the same developmental stage, postsynaptic site density in wt-hMeCP2-expressing neurons was similar to controls and increased at a rate higher than controls. Together, our in vivo studies support an early, cell-autonomous role for MeCP2 during the morphological differentiation of neurons and indicate that perturbations in MeCP2 gene dosage result in deficits in dendritic arborization that can be compensated, at least in part, by synaptic connectivity changes.
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Figure 1. Expression of MeCP2 in the developing Xenopus laevis visual system. (A) Endogenous expression of Xenopus MeCP2 mRNA in the
tectum and retina of stage 40 and stage 45 Xenopus tadpoles is shown by the RT-PCR reaction products. A single band of the expected molecular
weight was observed. Expression of the housekeeping gene x-GAPDH is also shown for comparison. DNA molecular weight markers are shown to the
left (M, in base pairs). (B) MeCP2 protein expression in the retina and optic tectum of Stage 40 tadpoles. Left panel: MeCP2 immunopositive cells
(green) are localized to the ganglion cell layer (gcl) and inner nuclear layer (inl) of the developing retina. The retinal synaptic layers are shown by the
immunostaining with an antibody to VAMPII (red). Right panel: Coronal section of a stage 40 tadpole at the level of the optic tectum shows MeCP2
expression in neurons (green) close to the tectal neuropil (n), which is visualized by VAMPII immunostaining (red). V = ventricle. Scale bar = 500 mm. (C,
D) Transfection with human wild-type hMeCP2 constructs was used to alter expression of MeCP2 in postmitotic Xenopus tectal neurons at the onset
of synaptic differentiation. C) Expression of wt-hMeCP2 was confirmed in triple transfected neurons co-expressing DsRed2, PSD-95-GFP and wthMeCP2
as illustrated here by the overlaid live confocal image (overlay), and the red (DsRed2) and green (PSD95-GFP) fluorescence as well as the
MeCP2 immunofluorescence (blue) after fixation. D) Tectal neuron transfected with DsRed2 and a wt-hMeCP2-IRES-GFP plasmid. Live confocal
imaging shows colocalization of DsRed2 (red) and GFP (green) in the nucleus, cell body, and primary dendrite. Retrospective immunostaining with an
antibody directed to human wild-type MeCP2 shows the localization of the MeCP2 protein to the nucleus and proximal portion of the primary
dendrite (blue). Scale bar for C, D= 10 mm.
doi:10.1371/journal.pone.0033153.g001
Figure 2. Expression of hMeCP2 influences tectal neuron dendritic branching. (A, B) Sample tectal neurons expressing DsRed2 (red)
together with GFP (green; IRES-GFP construct) from stage 45 Xenopus tadpoles illustrate the morphologies and dynamics of tectal neuron dendritic
branching over time. (C, D) Tectal neurons expressing DsRed2 (red) and wt-hMeCP2-IRES-GFP (green) in stage 45 Xenopus tadpoles illustrate the
effects of MeCP2 overexpression on dendritic morphology and branch dynamics. In these confocal projections, GFP expression (yellow; green and red
overlay) confirms the expression of wt-hMeCP2. The asterisks mark a primary dendrite and axons are demarcated by the arrows. Scale bar = 20 mm.
doi:10.1371/journal.pone.0033153.g002
Figure 3. Quantitative analysis of changes in dendritic arbor morphology induced by overexpression of wild-type hMeCP2. (A) Total
number of branches in tectal neurons of stage 45 Xenopus tadpoles at the initial observation time point, and 24 and 48 hours after initial imaging.
Note that control neurons increased their total number of branches over a 48 hr period, while wt-hMeCP2 expressing neurons had significantly fewer
branches and failed to increase branch number over time. (B) Total dendritic arbor length remained significantly lower in MeCP2 overexpressing
neurons, while control neurons increase their total dendritic arbor length in every 24 hr observation interval. (C) A relative measure of dendritic
segment length, calculated as the ratio of total arbor length by total branch number, shows that on average branches in hMeCP2-expressing neurons
are longer than in controls. (D) Left; Sholl analysis was used to determine the number of dendritic crossings in MeCP2 overexpressing and control
neurons at 0 hours as measure of dendrite morphology and length. Note that while the maximal extent of the dendritic arbor is similar in hMeCP2-
expressing neurons and controls, hMeCP2-expressing neurons have a more uniformly distributed pattern of dendrite lengths. Significance * p#0.05;
** p#0.005, ***p#0.001.
doi:10.1371/journal.pone.0033153.g003
Figure 4. hMeCP2-expressing neurons develop morphologically simple dendritic arbors. (A) The complexity of the dendritic arbors in
control neurons expressing DsRed2 and in neurons co-expressing DsRed2 and hMeCP2 is exemplified by the proportion of first, second, third and
fourth order branches, expressed as percent of their total branch number. Note that MeCP2 overexpressing neurons have proportionately more first
order branches but fewer third order branches. (B) Top; The Dendritic Complexity Index (DCI) provides an additional measure of dendritic
morphology. Bottom graph; The DCI value for MeCP2 expressing neurons was significantly lower than the value for control neurons at the initial
observation time point. Moreover, while control neurons significantly increased their DCI value by 48 h, DCI value for hMeCP2-expressing neurons did
not change over time. C, D) Branch order distribution at 0 and 48 hours for (C) control, and (D) hMeCP2-expressing neurons. Note the significant shift
in distribution of branches in control neurons, indicating an increase in complexity over time, while no change was observed over a 48 hour period in
neurons overexpressing MeCP2. Significance * p#0.05; ** p#0.005, ***p#0.001.
doi:10.1371/journal.pone.0033153.g004
Figure 5. Overexpression of MeCP2 decreases new branch formation in developing tectal neurons but does not interfere with the
stability of existing branches. The absolute (A) and relative (B) number of stabilized and newly added branches in MeCP2 overexpressing
neurons compared to controls are shown by the bar graphs. hMeCP2-expressing neurons added significantly fewer new branches than controls
during every 24 imaging period (04 h and 248 h, combined). As percentage, the number of dendritic branches stabilized over a 24 h period is
significantly higher in hMeCP2-expressing neurons than controls (B), although hMeCP2-expressing neurons had fewer dendritic branches overall (A,
absolute values; see also Fig. 3). Significance * p#0.05; ** p#0.005, ***p#0.001.
doi:10.1371/journal.pone.0033153.g005
Figure 6. Postsynaptic site differentiation in hMeCP2-expressing tectal neurons. Time lapse confocal images of representative control (A,
B) and hMeCP2-expressing (C, D) tectal neurons co-expressing DsRed2 (red) and PSD95-GFP (green) in stage 45 Xenopus tadpoles illustrate the
morphologies and distribution of PSD95-GFP postsynaptic specializations (yellow puncta; red and green overlap) on the dendritic arbors. (C, D) Tectal
neurons co-expressing wild-type hMeCP2 together with DsRed2 and PSD95-GFP show an increase in the density of postsynaptic clusters (yellow
puncta, arrowheads) over a 24 and 48 h observation period. Axons of tectal neurons are marked by white arrows. Expression of hMeCP2 was
confirmed by retrospective immunostaining as in Fig. 1D. Scale bar = 20 mm.
doi:10.1371/journal.pone.0033153.g006
Figure 7. Expression of hMeCP2 differentially influences dendritic branching and postsynaptic site differentiation. The effects of
MeCP2 overexpression on postsynaptic specializations in the tectal neuron dendritic arbors are shown by the bar graphs. (A) The absolute number of
PSD95-GFP postsynaptic clusters in the hMeCP2-expressing neurons is lower than in controls both at 0 and 24 h. (B) When normalized per unit arbor
length, the density of PSD95-GFP postsynaptic clusters is similar to controls at the initial observation time point but increases significantly more than
controls by 24 hours. (C) hMeCP2-expressing neurons increase their postsynaptic clusters number by approximately two-fold in 24 hours.
Significance * p#0.05; ** p#0.005, ***p#0.001.
doi:10.1371/journal.pone.0033153.g007
Vamp2 (vesicle-associated membrane protein 2 (synaptobrevin 2)) (RED) and Mecp2(methyl CpG binding protein 2 (Rett syndrome)) (GREEN) gene expression in Xenopus laevis embryos, NF stage 40, as assayed by immunofluorescence. Sagittal section of the eye, coronal section of the brain at the level of the optic tectum: Dorsal is up.
Mecp2 Ab1 staining in the Xenopus laevis NF stage 40 eye
Vamp2 Ab1 staining in Xenopus laevis NF stage 40 eye
Alsina,
Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF.
2001, Pubmed,
Xenbase
Alsina,
Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF.
2001,
Pubmed
,
Xenbase
Amir,
Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.
1999,
Pubmed
Armstrong,
Selective dendritic alterations in the cortex of Rett syndrome.
1995,
Pubmed
Armstrong,
The neuropathology of the Rett syndrome.
1992,
Pubmed
Asaka,
Hippocampal synaptic plasticity is impaired in the Mecp2-null mouse model of Rett syndrome.
2006,
Pubmed
Ballas,
Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology.
2009,
Pubmed
Balmer,
Elevated methyl-CpG-binding protein 2 expression is acquired during postnatal human brain development and is correlated with alternative polyadenylation.
2003,
Pubmed
Belichenko,
Evidence for both neuronal cell autonomous and nonautonomous effects of methyl-CpG-binding protein 2 in the cerebral cortex of female mice with Mecp2 mutation.
2009,
Pubmed
Belichenko,
Rett syndrome: 3-D confocal microscopy of cortical pyramidal dendrites and afferents.
1994,
Pubmed
Belichenko,
Widespread changes in dendritic and axonal morphology in Mecp2-mutant mouse models of Rett syndrome: evidence for disruption of neuronal networks.
2009,
Pubmed
Belichenko,
Studies on the 3-dimensional architecture of dendritic spines and varicosities in human cortex by confocal laser scanning microscopy and Lucifer yellow microinjections.
1995,
Pubmed
Ben-Shachar,
Mouse models of MeCP2 disorders share gene expression changes in the cerebellum and hypothalamus.
2009,
Pubmed
Boggio,
Synaptic determinants of rett syndrome.
2010,
Pubmed
Chahrour,
MeCP2, a key contributor to neurological disease, activates and represses transcription.
2008,
Pubmed
Chahrour,
The story of Rett syndrome: from clinic to neurobiology.
2007,
Pubmed
Chang,
The disease progression of Mecp2 mutant mice is affected by the level of BDNF expression.
2006,
Pubmed
Chao,
Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes.
2010,
Pubmed
Chao,
The yin and yang of MeCP2 phosphorylation.
2009,
Pubmed
Chao,
MeCP2 controls excitatory synaptic strength by regulating glutamatergic synapse number.
2007,
Pubmed
Chapleau,
Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations.
2009,
Pubmed
Chen,
Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice.
2001,
Pubmed
Chen,
Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2.
2003,
Pubmed
Cline,
Dendritic arbor development and synaptogenesis.
2001,
Pubmed
Cohen,
Expression of MeCP2 in olfactory receptor neurons is developmentally regulated and occurs before synaptogenesis.
2003,
Pubmed
Cohen-Cory,
The cellular patterns of BDNF and trkB expression suggest multiple roles for BDNF during Xenopus visual system development.
1996,
Pubmed
,
Xenbase
Cohen-Cory,
Neurotrophic regulation of retinal ganglion cell synaptic connectivity: from axons and dendrites to synapses.
2004,
Pubmed
,
Xenbase
Collins,
Mild overexpression of MeCP2 causes a progressive neurological disorder in mice.
2004,
Pubmed
Coverdale,
Differential expression of the methyl-cytosine binding protein 2 gene in embryonic and adult brain of zebrafish.
2004,
Pubmed
,
Xenbase
Dani,
Reduced cortical activity due to a shift in the balance between excitation and inhibition in a mouse model of Rett syndrome.
2005,
Pubmed
Degano,
MeCP2 deficiency disrupts axonal guidance, fasciculation, and targeting by altering Semaphorin 3F function.
2009,
Pubmed
Friez,
Recurrent infections, hypotonia, and mental retardation caused by duplication of MECP2 and adjacent region in Xq28.
2006,
Pubmed
Fukuda,
Delayed maturation of neuronal architecture and synaptogenesis in cerebral cortex of Mecp2-deficient mice.
2005,
Pubmed
Guy,
A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome.
2001,
Pubmed
Hite,
Recent advances in MeCP2 structure and function.
2009,
Pubmed
Hossain,
Dynamic morphometrics reveals contributions of dendritic growth cones and filopodia to dendritogenesis in the intact and awake embryonic brain.
2012,
Pubmed
,
Xenbase
Hu,
BDNF stabilizes synapses and maintains the structural complexity of optic axons in vivo.
2005,
Pubmed
,
Xenbase
Jugloff,
Increased dendritic complexity and axonal length in cultured mouse cortical neurons overexpressing methyl-CpG-binding protein MeCP2.
2005,
Pubmed
Jung,
The expression of methyl CpG binding factor MeCP2 correlates with cellular differentiation in the developing rat brain and in cultured cells.
2003,
Pubmed
Kishi,
MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions.
2004,
Pubmed
,
Xenbase
Kishi,
MeCP2 functions largely cell-autonomously, but also non-cell-autonomously, in neuronal maturation and dendritic arborization of cortical pyramidal neurons.
2010,
Pubmed
Kline,
Exogenous brain-derived neurotrophic factor rescues synaptic dysfunction in Mecp2-null mice.
2010,
Pubmed
Larimore,
Bdnf overexpression in hippocampal neurons prevents dendritic atrophy caused by Rett-associated MECP2 mutations.
2009,
Pubmed
Lugtenberg,
Chromosomal copy number changes in patients with non-syndromic X linked mental retardation detected by array CGH.
2006,
Pubmed
Marshak,
Cell-autonomous TrkB signaling in presynaptic retinal ganglion cells mediates axon arbor growth and synapse maturation during the establishment of retinotectal synaptic connectivity.
2007,
Pubmed
,
Xenbase
Martinowich,
DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation.
2003,
Pubmed
Moretti,
Learning and memory and synaptic plasticity are impaired in a mouse model of Rett syndrome.
2006,
Pubmed
Mullaney,
Developmental expression of methyl-CpG binding protein 2 is dynamically regulated in the rodent brain.
2004,
Pubmed
Nelson,
MeCP2-dependent transcriptional repression regulates excitatory neurotransmission.
2006,
Pubmed
Niell,
Live optical imaging of nervous system development.
2004,
Pubmed
Samaco,
Loss of MeCP2 in aminergic neurons causes cell-autonomous defects in neurotransmitter synthesis and specific behavioral abnormalities.
2009,
Pubmed
Samaco,
Complexities of Rett syndrome and MeCP2.
2011,
Pubmed
Sanchez,
BDNF increases synapse density in dendrites of developing tectal neurons in vivo.
2006,
Pubmed
,
Xenbase
Shahbazian,
Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3.
2002,
Pubmed
Shepherd,
Synaptic microcircuit dysfunction in genetic models of neurodevelopmental disorders: focus on Mecp2 and Met.
2011,
Pubmed
Smrt,
Mecp2 deficiency leads to delayed maturation and altered gene expression in hippocampal neurons.
2007,
Pubmed
Stancheva,
A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in Xenopus embryos.
2003,
Pubmed
,
Xenbase
Tao,
Activity-dependent matching of excitatory and inhibitory inputs during refinement of visual receptive fields.
2005,
Pubmed
,
Xenbase
Tripodi,
Structural homeostasis: compensatory adjustments of dendritic arbor geometry in response to variations of synaptic input.
2008,
Pubmed
Van Esch,
Duplication of the MECP2 region is a frequent cause of severe mental retardation and progressive neurological symptoms in males.
2005,
Pubmed
Zhou,
Brain-specific phosphorylation of MeCP2 regulates activity-dependent Bdnf transcription, dendritic growth, and spine maturation.
2006,
Pubmed
del Gaudio,
Increased MECP2 gene copy number as the result of genomic duplication in neurodevelopmentally delayed males.
2006,
Pubmed
