Evolutionary Plasticity of Habenular Asymmetry with a Conserved Efferent Connectivity Pattern

The vertebrate habenulae (Hb) is an evolutionary conserved dorsal diencephalic nuclear complex that relays information from limbic and striatal forebrain regions to the ventral midbrain. One key feature of this bilateral nucleus is the presence of left-right differences in size, cytoarchitecture, connectivity, neurochemistry and/or gene expression. In teleosts, habenular asymmetry has been associated with preferential innervation of left-right habenular efferents into dorso-ventral domains of the midbrain interpeduncular nucleus (IPN). However, the degree of conservation of this trait and its relation to the structural asymmetries of the Hb are currently unknown. To address these questions, we performed the first systematic comparative analysis of structural and connectional asymmetries of the Hb in teleosts. We found striking inter-species variability in the overall shape and cytoarchitecture of the Hb, and in the frequency, strength and to a lesser degree, laterality of habenular volume at the population level. Directional asymmetry of the Hb was either to the left in D. rerio, E. bicolor, O. latipes, P. reticulata, B. splendens, or to the right in F. gardneri females. In contrast, asymmetry was absent in P. scalare and F. gardneri males at the population level, although in these species the Hb displayed volumetric asymmetries at the individual level. Inter-species variability was more pronounced across orders than within a single order, and coexisted with an overall conserved laterotopic representation of left-right habenular efferents into dorso-ventral domains of the IPN. These results suggest that the circuit design involving the Hb of teleosts promotes structural flexibility depending on developmental, cognitive and/or behavioural pressures, without affecting the main midbrain connectivity output, thus unveiling a key conserved role of this connectivity trait in the function of the circuit. We propose that ontogenic plasticity in habenular morphogenesis underlies the observed inter-species variations in habenular asymmetric morphology

Cell-Autonomous Alterations in Dendritic Arbor Morphology and Connectivity Induced by Overexpression of MeCP2 in Xenopus Central Neurons In Vivo

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

Cell-Autonomous Alterations in Dendritic Arbor Morphology and Connectivity Induced by Overexpression of MeCP2 in Xenopus Central Neurons In Vivo

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

Asymmetry and laterality index of the habenulae in teleosts.

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

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

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

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

Comparative analysis of volumetric asymmetry in the habenulae 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>. For each species, the corresponding panels from columns B to F are aligned horizontally. (B) Schematics of habenular cytoarchitecture obtained from the cresyl-violet stained coronal sections of the Hb shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035329#pone-0035329-g001" target="_blank">Figure 1</a>. (C) Volumetric models of the Hb as seen dorsally, with anterior to the top. Volumes of the left and right Hb have been differentially coloured in red and green, respectively. (D) Colour code diagram indicating the presence of statistically significant laterality of habenular volume at the population level ([R-L]≠0). For each box, the presence of left- or right- directional asymmetries, and the corresponding p values, have been coloured according to the colour scale given below. Vertical lines and asterisks placed on the left side of some pairs of boxes indicate sex-specific significant differences in habenular asymmetry (* = p<0.05; ** = p<0.01). (E) Box plots indicating the scores of habenular Laterality Index for each species and sex. Positive and negative values indicate right- and a left- sided laterality of habenular volume, respectively. Values reveal the strength and directionality of habenular asymmetry at the population level. The vertical line with asterisks placed indicates sex-specific significant differences in Laterality Index (** = p<0.01). (F) Box plots indicating the absolute values of Laterality Index (abs-LI) for each species and sex, which reveal the strength of habenular asymmetry at the individual level. For each plot shown in E and F, the box depicts the interquartile range containing 50% of the data around the median (vertical line inside the box), and the whisker depicts maximum and minimum vales. Abbreviations: A (anterior), Asym (asymmetry), Cyto (cytoarchitecture), D (dorsal), F (females), L (left), M (males), P (posterior), R (right), V (ventral). Scale bar: 100 µm for column B, and 200 µm for column C.</p