. Summary

Research Projects / BRAIN ASYMMETRY

Despite the relevance of asymmetry in normal and affected brain function, the mechanism by which it is established during development has been largely elusive.
LEO's scientific approach uses the epithalamus of zebrafish and medaka as a main model system to address the ontogenic mechanisms of brain asymmetry, applying a bottom-up (gene to behaviour) comparative analysis.

Asymmetry is a conserved and fundamental feature of the brain that is thought to enhance efficiency in information processing. Recently, we have gained insights into the developmental basis of brain asymmetry owing to the use of genetic model organisms that allow a comprehensive bottom-up (gene to behaviour) approach (see Concha 2004 [pdf]). In vertebrates, one such organism is zebrafish as it offers several practical advantages. First, it is a well-established genetic system in which to explore the role of genes at the various levels of brain asymmetry organisation. Second, the transparency of embryos and larvae that exhibit external development allows the visualisation of neuronal and axonal morphogenesis within asymmetric circuits in vivo. Third, the feasibility to assess neuronal activity and behaviour allows the establishment of operational links between lower (genetic, structural) and upper (functional, behavioural) levels of brain lateralisation. Finally, zebrafish offers a unique opportunity to dissect conserved and species-specific mechanisms of brain asymmetry through comparative developmental studies between related teleost species.

The best-studied example of asymmetry in the zebrafish brain is observed in the epithalamus, a region of the dorsal diencephalon containing the pineal complex and habenulae. The pineal complex is a photoreceptive neuroendocrine cell group involved in the physiology of circadian rhythms whereas the habenulae is a paired bilateral structure that serves as a relay station linking the limbic forebrain and the ventral midbrain (see review of the epithalamus in Concha and Wilson 2001 [pdf]).

The pineal complex is formed by pineal (pi) and parapineal (pp) organs, and the most conspicuous asymmetry corresponds to the left-sided asymmetric positioning and connectivity of the parapineal organ. During embryogenesis, this neuronal structure stems from the dorsal midline and migrates asymmetrically to the left side. On the other hand, asymmetries between left (lHb) and right (rHb) habenular nuclei are observed at gene and protein expression levels, in the cytoarchitecture and ratio of habenular sub-nuclei, and in the morphology and connectivity of habenular projection neurons. Typically, projections from left and right habenular neurons segregate in a dorso (d) to ventral (v) manner in the interpeduncular nucleus (IPN) of the ventral midbrain.


Epithalamic asymmetry belongs to the class of directional or population-level asymmetry, as it is inherited and most individuals are asymmetrical in the same direction within the population. Indeed, parapineal and habenular asymmetries are directed to the left side in more than 95% of the wild type zebrafish population. During embryogenesis, the two main aspects of directional asymmetry, asymmetry per se (structural differences between left and right sides at the individual level) and laterality (directionality of asymmetry at a population level) are controlled by independent genetic mechanisms (reviewed in Concha et al 2009 [pdf]). Asymmetry is controlled by an Fgf-dependent bi-stable cell migratory event that directs parapineal organ migration to either left or right in an anti-symmetric (random) manner (Regan et al 2009 [pdf]). Laterality, on the other hand, is controlled by the left-sided asymmetric activation of Nodal signalling in the epithalamus (Concha et al 2000 [pdf]). Importantly, laterality of epithalamic asymmetry is coupled to laterality of visceral asymmetry in contrast to other structural and functional asymmetries of the vertebrate brain, e.g. asymmetries associated to speech and handedness. This indicates that asymmetries controlled by independent mechanisms co-exist in the vertebrate brain.

Asymmetries of the parapineal organ and habenulae develop sequentially and their interactions mutually enhance individual asymmetries and the final configuration of lateralised circuits. This observation suggests that the ontogenic mechanisms of epithalamic asymmetry organise into distinct developmental modules that arrange in a causally dependent sequential manner (Concha et al 2009). Each developmental module exhibits distinct inputs and internal processes that allow a core function (output) to become robust to change.

The visceral laterality module gathers antero-posterior and dorso-ventral positional information (input) and involves a cilia-dependent mechanism of directional extracellular fluid flow in the Kupffer’s vesicle (internal process) that result in asymmetric Nodal signalling within the left lateral plate mesoderm (output) (A). Such a signal acts as an input for the epithalamic laterality module to couple visceral and brain laterality. This occurs through the modulation of a Wnt/ß-catenin Six3-dependent repressive interaction in the epithalamus (internal process), which produces left-sided Nodal signalling (outcome) (B). Unilateral Nodal signalling then biases the activity of the parapineal anti-symmetry module. This involves the modulation of an Fgf8-dependent bi-stable cell migratory event (internal process) that generates left-sided asymmetric migration of the parapineal organ (output) (C). An asymmetric parapineal organ exerts a local amplification influence (input) on the activity the habenular asymmetric morphogenesis module to enhance pre-existing left-right asymmetries (internal process) (D). Parapineal-mediated amplification results in distinct morphology, expression of guidance cues and ratio of lateral and medial habenular projection neurons in the left and right habenula (output) (D). Such asymmetries result in the segregation of axonal terminals from lateral (primarily left) and medial (fundamentally right) habenular projection neurons in dorsal and ventral regions of the IPN, respectively. This is the input of the IPN laterotopy module whose primary function is to convey lateralised epithalamic neuronal activity into bilateral motor circuitry on both sides of the midline (output) (E).


Recent comparative surveys reveal a striking conservation of epithalamic asymmetry among a wide range of vertebrate species (reviewed in Concha and Wilson 2001) [pdf]). Current knowledge is consistent with the idea that the epithalamic region was present in chordates prior to the origin of vertebrates. However, when and how asymmetry first appeared during evolution remains unclear. Our current model proposes that the pineal complex originally consisted of bilateral structures containing both pineal and parapineal components (see Fig. 7 in Concha et al 2009 [pdf]). Both sides of the pineal complex then fused at the midline and separated along the antero-posterior axis to give rise to pineal and parapineal organs. Habenular asymmetries possibly involving stochastic differences of a bilaterally symmetric signal may have also appeared at this stage of evolution. The acquisition of ontogenic mechanisms to induce the formation of a compact parapineal organ as an independent structure within the epithalamus and the activation of cell motility were possibly at the origin of a bi-stable cell morphogenetic event that directed parapineal migration to either right or left in an anti-symmetric manner. A laterality module involving Nodal-dependent signalling was then co-opted from the process of visceral asymmetry to bias the bi-stable system and impose consistent laterality of parapineal migration at a population level. In a final step, the asymmetric positioning of the parapineal organ in close proximity to the left habenula may have allowed the establishment of positive or negative local influences on left habenular development to regulate cellular processes such as neurogenesis and/or proliferation. Such influence manifested as either increased or decreased complexity of left habenular cytoarchitecture, sub-nuclear organisation, and/or target selectivity of projection neurons, which resulted in enhanced left-right asymmetries. As parapineal organs have been observed only in lampreys, teleosts and lizards (and perhaps also in frogs) and because they share fundamental embryological, topological and hodological features, it seems likely that this structure was present in the ancestral chordate/vertebrate and was subsequently lost in several vertebrate lineages independently. Importantly, loss of the parapineal organ in these vertebrate groups uncovered pre-existing parapineal-independent habenular asymmetries. It is though intriguing that laterality of these asymmetries shows a large degree of variation among vertebrates. This observation suggests that the mechanisms involved in the establishment of early parapineal-independent habenular asymmetries produced anti-symmetric (left or right) phenotypes that were independently fixed during the evolution of different vertebrate lineages.

Scientific Approach

LEO uses the epithalamus of zebrafish as a model system to address four main aspects of the ontogeny of brain asymmetry: [GENE . MORPH . EVO-DEVO . BIOMED]


We have recently shown that an Fgf-dependent bi-stable cell migratory event is involved in setting-up asymmetry per se (Regan et al 2009 [pdf]) whereas a bias imposed by asymmetric Nodal signalling controls the laterality of asymmetry (Concha et al 2000 [pdf]). How other aspects of asymmetry, such as asymmetric morphogenesis, are genetically controlled remains unknown. At LEO we explore the role of genetic pathways during different phases of asymmetric morphogenesis following candidate gene approaches. Current research focuses on (i) the role of Fgf and chemokine signalling in the development of the parapineal-habenular-IPN axis, and (ii) the search of novel genes with asymmetric expression in the zebrafish brain using subtractive hybridisation technology. The figure shows the expression (via in situ hybridisation) of a novel gene with asymmetric expression in the habenulae, recently isolated from a reverse genetic screen.
LEO members: Alicia Colombo (NECI-lab), Néstor Guerrero, Lorena Armijo, Margarita Meynard, Karina Palma.


Asymmetric morphogenesis of the epithalamus involves the left-sided migration of the parapineal nucleus (Concha et al 2003 [pdf]), the development of asymmetric habenular neuropil (Concha et al 2000 [pdf]) and the segregation of habenular efferent connectivity along the dorso-ventral axis of the IPN (Aizawa et al 2002 [pdf]). LEO has established a "Mutual Enhancement" collaborative relationship [see definition] with the Laboratory of Scientific Image Analysis (SCIAN-lab) to apply a multi-scale morpho-topological approach using in vivo microscopy to study in detail the morphogenetic transformations of asymmetric structures at supra-cellular, cellular and sub-cellular levels (Hartel et al 2007 [pdf]). Current research focuses on (i) parapineal nucleus formation and migration, and (ii) the acquisition of neuronal morphology and branching during habenular neuropil formation. The figure shows examples of morpho-topological analyses of parapineal nucleus morphogenesis.
LEO members: Carmen Lemus, Karina Palma, Lorena Armijo, Margarita Meynard.


To unveil those aspects that represent the backbone of epithalamic asymmetry and those subjected to evolutionary variation, LEO is perfoming a systematic comparative analyses of epithalamic asymmetry development among teleosts. Recent work revealed an overall conservation of asymmetry along the parapineal-habenular-IPN axis between zebrafish and medaka, which is accompanied by heterotopic changes in the topology of parapineal connectivity and heterochronic shifts in the timing of developmental events underlying the establishment of asymmetry (Signore et al 2009 [pdf]). Current research focus on (i) the mechanisms of evolved variations in the timing of ontogenic events of asymmetry between zebrafish and medaka, and (ii) the morpho-topological configuration of habenular asymmetry among teleosts. The figure shows the morphology of the pineal complex in transgenic medaka and zebrafish.
LEO members: Iskra Signore, Aldo Villalón, Mauro Sepúlveda, Geraldine Vásquez.


Compromised asymmetry has been associated to several human neuropathologies including schizophrenia, autism and neuronal degenerative diseases. In addition, components of the epithalamus are immersed in an evolutionary conserved circuit involved in limbic-system related responses, which has been implicated in the control of negative reward signals and origin of neuropsychiatric disorders. Altogether, this information underscores the relevance of adressing the function of asymmetry in this neuronal circuit. LEO is currently beginning to address the biomedical implication of epithalamic symmetry and asymmetry following two main research strategies: (i) exploring the existance of morpho-topological asymmetries in the habenular complex of humans, and (ii) developing in vivo models to address the function of epithalamic asymmetry in zebrafish larvae. The figure shows a 3D reconstruction of the human habenular complex.
LEO members: Felipe Fredes, Eugenia Díaz, Daniela Bravo, Ximena Rojas, Mauro Sepúlveda.



. Aizawa H, Bianco IH, Hamaoka T, Miyashita T, Uemura O, Concha ML, et al. Laterotopic representation of left-right information onto the dorso-ventral axis of a zebrafish midbrain target nucleus. Curr Biol 2005;15:238-43. [pdf]
. Concha ML, Burdine RD, Russell C, Schier AF, Wilson SW. A nodal signaling pathway regulates the laterality of neuroanatomical asymmetries in the zebrafish forebrain. Neuron 2000;28:399-409. [pdf]
. Concha ML, Wilson SW. Asymmetry in the epithalamus of vertebrates. J Anat 2001;199:63-84. [pdf]
. Concha ML, Russell C, Regan JC, Tawk M, Sidi S, Gilmour DT, et al. Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality. Neuron 2003;39:423-38. [pdf]
. Concha ML. The dorsal diencephalic conduction system of zebrafish as a model of vertebrate brain lateralisation. Neuroreport 2004;15:1843-6. [pdf]
. Concha M.L., Signore I.A., and Colombo A. (2009) Semin Cell Dev Biol 20, 498-509. [pdf]
. Härtel S., Jara J., Lemus C.G., and Concha M.L. (2007) In Computational Modelling of Objects Represented in Images. Fundamentals, Methods and Applications. Ed. João Manuel Tavares & Jorge Nata, Taylor and Francis Group, ISBN: 9780415433495, pp: 215-22. [pdf]
. Regan JC, Concha ML, Roussigne M, Russell C, Wilson SW. An Fgf8-dependent bi-stable cell migratory event establishes CNS asymmetry. Neuron 61, 27-34. [pdf]
. Signore IA, Guerrero N, Loosli F, Colombo A, Villalon A, Wittbrodt J, Concha ML. Zebrafish and medaka: model organisms for a comparative developmental approach of brain asymmetry. Philos Trans R Soc Lond B Biol Sci 364, 991-1004. [pdf]

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