Quantitative analysis of changes in dendritic arbor morphology induced by overexpression of wild-type hMeCP2.

<p>(<b>A</b>) Total number of branches in tectal neurons of stage 45 <i>Xenopus</i> 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>B</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. (<b>C</b>) 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. (<b>D</b>) <i>Left</i>; 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.</p

Expression of MeCP2 in the developing <i>Xenopus laevis</i> visual system.

<p>(<b>A</b>) Endogenous expression of <i>Xenopus</i> MeCP2 mRNA in the tectum and retina of stage 40 and stage 45 <i>Xenopus</i> tadpoles is shown by the RT-PCR reaction products. A single band of the expected molecular weight was observed. Expression of the housekeeping gene <i>x</i>-GAPDH is also shown for comparison. DNA molecular weight markers are shown to the left (M, in base pairs). (<b>B</b>) MeCP2 protein expression in the retina and optic tectum of Stage 40 tadpoles. <i>Left panel:</i> MeCP2 immunopositive cells (green) are localized to the ganglion cell layer (<i>gcl</i>) and inner nuclear layer (<i>inl</i>) of the developing retina. The retinal synaptic layers are shown by the immunostaining with an antibody to VAMPII (<i>red</i>). <i>Right panel:</i> Coronal section of a stage 40 tadpole at the level of the optic tectum shows MeCP2 expression in neurons (<i>green</i>) close to the tectal neuropil (<i>n</i>), which is visualized by VAMPII immunostaining (<i>red</i>). V = ventricle. Scale bar = 500 µm. (<b>C, D</b>) Transfection with human wild-type hMeCP2 constructs was used to alter expression of MeCP2 in postmitotic <i>Xenopus</i> tectal neurons at the onset of synaptic differentiation. <b>C</b>) Expression of wt-hMeCP2 was confirmed in triple transfected neurons co-expressing DsRed2, PSD-95-GFP and wt-hMeCP2 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. <b>D</b>) Tectal neuron transfected with DsRed2 and a wt-hMeCP2-IRES-GFP plasmid. Live confocal imaging shows colocalization of DsRed2 (<i>red</i>) and GFP (<i>green</i>) 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 (<i>blue</i>). Scale bar for C, D = 10 µm.</p

hMeCP2-expressing neurons develop morphologically simple dendritic arbors.

<p>(<b>A</b>) 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>B</b>) <i>Top</i>; The Dendritic Complexity Index (DCI) provides an additional measure of dendritic morphology. <i>Bottom graph</i>; 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. <b>C, D</b>) Branch order distribution at 0 and 48 hours for (<b><i>C</i></b>) control, and (<b><i>D</i></b>) 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.</p

Overexpression of MeCP2 decreases new branch formation in developing tectal neurons but does not interfere with the stability of existing branches.

<p>The absolute (<b>A</b>) and relative (<b>B</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 (0–24 h and 24–48 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><i>B</i></b>), although hMeCP2-expressing neurons had fewer dendritic branches overall (<b><i>A</i></b>, absolute values; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033153#pone-0033153-g003" target="_blank"><b><i>Fig. 3</i></b></a>). Significance * p≤0.05; ** p≤0.005, ***p≤0.001.</p

Expression of hMeCP2 influences tectal neuron dendritic branching.

<p>(<b>A, B</b>) Sample tectal neurons expressing DsRed2 (<i>red</i>) together with GFP (<i>green</i>; IRES-GFP construct) from stage 45 <i>Xenopus</i> tadpoles illustrate the morphologies and dynamics of tectal neuron dendritic branching over time. (<b>C, D</b>) Tectal neurons expressing DsRed2 (<i>red</i>) and wt-hMeCP2-IRES-GFP (<i>green</i>) in stage 45 <i>Xenopus</i> tadpoles illustrate the effects of MeCP2 overexpression on dendritic morphology and branch dynamics. In these confocal projections, GFP expression (<i>yellow</i>; 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 µm.</p

Expression of hMeCP2 differentially influences dendritic branching and postsynaptic site differentiation.

<p>The effects of MeCP2 overexpression on postsynaptic specializations in the tectal neuron dendritic arbors are shown by the bar graphs. (<b>A</b>) 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>B</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. (<b>C</b>) 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.</p

Postsynaptic site differentiation in hMeCP2-expressing tectal neurons.

<p>Time lapse confocal images of representative control (<b><i>A, B</i></b>) and hMeCP2-expressing (<b><i>C, D</i></b>) tectal neurons co-expressing DsRed2 (<i>red</i>) and PSD95-GFP (<i>green</i>) in stage 45 <i>Xenopus</i> tadpoles illustrate the morphologies and distribution of PSD95-GFP postsynaptic specializations (<i>yellow puncta</i>; red and green overlap) on the dendritic arbors. (<b>C, D</b>) Tectal neurons co-expressing wild-type hMeCP2 together with DsRed2 and PSD95-GFP show an increase in the density of postsynaptic clusters (<i>yellow puncta</i>, 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033153#pone-0033153-g001" target="_blank">Fig. 1D</a>. Scale bar = 20 µm.</p