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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
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