Asymmetry and laterality index of the habenulae in teleosts.

<p>Abbreviations: L (Left), Max (maximum value), Min (minimum value), R (Right).</p

Relationship between habenular volume and laterality Index.

<p>(A–D) Plots showing the relation between habenular volume (rHb+lHb, in mm³×10⁻³) and asymmetry (rHb - lHb, in mm³×10⁻⁴) in different species of teleosts. Data corresponding to different species have been presented in two rows, each representing sex (female = top; male = bottom), and grouped into two columns according to the size of individuals (left = smaller fish; right = larger fish). Groups of dots sharing the same colour correspond to individuals of a single species, and the line of equivalent colour depicts the linear regression of that group. The abbreviation for each species is given on either left or right sides of the regression line, according to the code given in E. (E) Pearson's correlation coefficient (r) and p values (in parenthesis) for each species and sex. The asterisk indicates the presence of statistically significant correlation between habenular volume and asymmetry (p<0.05).</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

Cytoarchitectonic organisation of the habenulae in teleosts.

<p>(A) Drawings of adult male individuals belonging to different teleost species, placed in the context of a cladogram of the teleost lineage according to Nelson <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035329#pone.0035329-Nelson1" target="_blank">[28]</a>. (B) Schematic representation of a teleost brain (e.g. D rerio), showing the location and orientation of histological sections shown in C–I. (C–I) Photomicrographs of cresyl-violet stained 10 µm-thick coronal sections taken at a midpoint between rostral and caudal ends of the Hb as shown in B. Each panel corresponds to a single species, as indicated in the letter code of the left diagram. C′ is a magnification of the square region depicted in C. Dorsal is to the top, and left is to the left. Arrowheads point to the subhabenular sulcus. Asterisks indicate the position of the dorsal-most neuropil region of the dorsal habenulae that is surrounded by a shell of cell bodies in some species. Abbreviations: A (anterior), D (dorsal), dHb (dorsal Hb), L (left), P (posterior), R (right), TeO (Optic Tectum), V (ventral), vHb (ventral Hb). Scale bars: 50 µm.</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

Comparative distribution of left and right habenular efferents in the interpeduncular nucleus of teleosts.

<p>(A) Drawings of adult male individuals belonging to different teleost species, placed in the context of a cladogram of the teleost lineage according to Nelson <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035329#pone.0035329-Nelson1" target="_blank">[28]</a>. (B) Schematic representation of a teleost brain (e.g. D rerio), showing the procedure of differential dye labelling in left (DiD, red) and right (DiO, green) Hb, and the location and orientation of the histological sections shown in B′, and C–I. (B′) Schematic drawing of a coronal section at the level of the IPN in a teleost brain (e.g. D rerio), showing dorsal and ventral aspects of the IPN in red and green, respectively. (C–I) Confocal microscopy images of 100 µm-thick vibratome sections taken according to B′, with dorsal to the top. The boundaries of dorsal (dIPN) and ventral (vIPN) IPN domains have been depicted with dashed lines. Panels C to I correspond to efferents labelled after placing crystals of DiD in the left Hb. Panels C′ to I′ correspond to efferents labelled after placing crystals of DiO in the right Hb. Abbreviations: A (anterior), D (dorsal), P (posterior), TeO (Optic Tectum), V (ventral). Scale bars: 100 µm.</p

Sex-specific differences in volume, asymmetry and laterality index of the habenulae in teleosts.

<p>Abbreviations: male>female [m], female>male [f].</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

Social vs Non-social differences in the laterality index of the habenulae in teleosts.

<p>Social vs Non-social differences in the laterality index of the habenulae in teleosts.</p