How do bilaterally equivalent structures in the forebrain become left-right asymmetric?

This publication summary relates to the paper:

Cachd1 interacts with Wnt receptors and regulates neuronal asymmetry in the zebrafish brain (Powell et al., 2024)

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The human brain is divided into left and right hemispheres that superficially look like mirror copies of each other. However, there are differences between the sides that ultimately affect the types of neural circuits formed, the function of those circuits, and our overall behaviour. Many of these lateralised differences are also found in other animals and appear to have been conserved in evolution. One example of such conservation is the habenulae: two small brain nuclei, one present on either side, that act as a relay station to integrate sensory information and then pass a signal on to other circuits that control some forms of motor behaviour. We study the habenulae in zebrafish because there are easy to observe left-right asymmetries in the translucent embryo, such as size and shape, gene expression and circuit inputs and outputs (see figure below, A-C: compare wildtype panels on the left with mutant panels on the right). How those differences arise, and what affect they have on behaviour, is still not very well understood. In the Wilson lab, we look at how asymmetries are established in the brain during embryonic development by exploring what genes are involved in the process, what effect those genes have on the structure of the habenulae, and finally, how the zebrafish behave.

We have recently discovered that a gene called cachd1 plays a very important early role in establishing the difference between the left and right habenulae: when it is mutated, the right habenula develops into an almost mirror copy of the left habenula (see figure below, A and B). We have learnt that cachd1 does this, at least in part, by controlling the time at which the habenula neurons are born. Normally, more neurons are born early on the left-hand side than the right, and this affects the character of the mature habenulae (see figure below, D, left panel). When cachd1 does not function, more neurons are born early on both sides (see figure below, D, right panel), leading to mature habenula that look alike, so-called “double left” habenulae. This in turn affects the type of sensory circuits that can connect to the habenula, and how the habenulae connect to downstream circuits (see figure below, C).

We believe that cachd1 is able to control the timing of neurons by modulating a key developmental signalling pathway called the Wnt pathway. Wnt is a secreted protein that simultaneously binds to two receptor proteins (Frizzled and LRP6) on the surface of cells. This interaction sets off a chain reaction of events inside the cell, passing information to the nucleus allowing the cell to react to its environment. Wnt signalling is involved in lots of different developmental processes in the embryo, typically (but not exclusively) to do with controlling the proliferation of cells. Similar to the Wnt protein, Cachd1 protein is able to physically bind to the Frizzled and LRP6 receptors (see figure below, E), potentially competing with the signal to alter the output of the pathway; in this case, the time at which a habenula neuron is born. Consistent with this, mutation of lrp6 also causes “double left” habenulae to develop (see figure below, F).

Interestingly, cachd1 isn’t limited to either side of the brain in the embryo, so how does it control asymmetry? We believe that the function of cachd1 is suppressed by a signal from the parapineal – a small population of neurons that migrates to the left-hand side and contacts the left habenula (see [Ingrid’s paper summary]). When the parapineal is ablated, or cannot function properly due to mutation, the left habenula develops as a mirror copy of the right habenula, so-called “double right” habenulae (see figure below, G, left panel). However, when the parapineal is ablated in embryos lacking cachd1 function, “double left” habenulae develop (see figure below, G, right panel). This means that the parapineal must inhibit cachd1 in order for the left habenula to form in the embryo.

This is an important piece of the puzzle, but lots remain to be discovered. We are particularly interested in learning about the consequences of the cachd1 mutation on behaviour, as well as developing a better understanding of how the Cachd1 protein affects Wnt signalling and what the identity of the antagonising parapineal signal is. Furthermore, our discovery of the function of cachd1 will help us uncover more genes that play roles in determining left-right asymmetry.

Loss of function of cachd1 disrupts asymmetry of the habenulae. Mutation of cachd1 (right panels A-D, G; compare to wildtype, left panels) affects asymmetry of gene expression (A) and neuron composition (B; neurons in red, Cachd1 protein in cyan) of the habenulae (dashed box in A), as well as connections of the habenula neurons to other brain regions (C; left side neurons in magenta, right side neurons in green). Neurons are born early in cachd1 mutant embryos on both sides (D), with an increased proportion of left-character neurons on the right (magenta). When neurons are born in the habenulae depends (in part) on Wnt signalling through Frizzled (FZD) and Lrp6 co-receptors. Cachd1 protein can bind to these simultaneously, shown in the crystal structure of the complex formed by the three proteins (E). Mutation of lrp6 also affects asymmetry in the habenula (F), consistent with its interaction with Cachd1. When the parapineal is ablated in a wildtype embryo, both left and right habenula express a gene characteristic of the right habenula (G, left panel, green; pineal gland shown in white); this suggests that a signal from the parapineal is needed to break asymmetry. When it is ablated in the cachd1 mutant however, “double left” habenulae form; this suggests that cachd1 is needed to form the right habenulae, and must be suppressed by the parapineal signal on the left.

For the whole story of cachd1 check out our preprint on biorxiv.

By Gareth Powell

The power of the parapineal in brain asymmetry development

A summary of the paper “ Sox1a mediates the ability of the parapineal to impart habenular left-right asymmetry” eLife 2019;8:e47376 DOI: 10.7554/eLife.47376

The transparent larval zebrafish is excellent for studying brain development, allowing whole-embryo imaging and relatively simple genetic manipulation. Using these advantages, we study how the left and right side of the brain become different from each other (i.e. asymmetric) during embryogenesis.

The pineal is midline brain structure that contributes to the regulation of circadian rhythms in vertebrates. In zebrafish, the pineal is accompanied by an exclusively left-sided parapineal nucleus, function of which is not quite clear. It is known, however, that during embryonic development the parapineal emerges from the pineal and migrates to the left side (Figure 1). On this journey the parapineal cells instruct adjacent habenular neurons on the left side of the epithalamus (dorsal part of the thalamus) to become different from equivalent neurons on the right with regards to molecular properties and neuronal connectivity. In other words, the parapineal determines the development of left-right asymmetries in the habenulae. Eliminating or changing the side of the parapineal therefore leads to loss or reversal of habenular asymmetries, respectively (Figure 1).

Figure 1: The parapineal emerges from the pineal during zebrafish embryogenesis, as shown by confocal images of a transgenic line with GFP in the pineal complex (top pictures). The pineal complex lies at the midline of the epithalamus, flanked by left (green on the scheme) and right (magenta on the scheme) habenulae. The habenulae are innervated by the parapineal and themselves send neuronal projections to the midbrain interpeduncular nucleus (IPN). The side of the parapineal, on the left in wild-type (wt), determines which habenula acquires a left-type identity projecting to the dorsal part of the IPN (dIPN).

We show that during embryogenesis the parapineal instructs the cells in the left habenula to initiate neurogenesis earlier compared to the right habenula. The parapineal is also required at later stages for the establishment neuronal connectivity specific to the left habenula.

Upon precise laser-ablation of the embryonic parapineal cells prior to their migration to the left side, both habenulae exhibit right habenula characteristics at larval stages. A similar phenotype can be observed upon mutating the sox1a gene, normally active in the parapineal. In sox1a mutants the parapineal forms properly, but the habenulae develop as if there was no parapineal. Finally, transplanting only a few parapineal cells to the right side of the embryonic brain induces left-sided character in neurons within the right habenula. The results of these experiments are schematically summarised in Figure 2.

Figure 2: One of the mechanisms leading to left-right asymmetries in the zebrafish habenulae is the different timing of neurogenesis on each side. In sox1a-/- mutants and upon parapineal ablation the early onset of neurogenesis in the left habenula is delayed and the habenulae acquire a double-right character. Hence, in the absence of the left-sided parapineal or upon loss of sox1a-/- in the parapineal the left habenula becomes identical to the right habenula. Conversely, transplantation of external parapineal cells to the right side induces left-habenula characteristics in the right habenula.

The development of the zebrafish epithalamus serves as a great example of a genetically determined mechanism for brain asymmetry formation. The emergence of a parapineal nucleus in the pineal complex during evolution has in turn led to asymmetries in the habenulae, ensuring that appropriate lateralised character is propagated within left and right-sided circuitry. As we and others have previously shown, the asymmetric neuroanatomy translates into asymmetric function of the habenulae and lateralised behaviour of the fish. Hence, from studies in zebrafish a clearer picture is forming on how and why brain asymmetries emerge in largely­ bilaterally symmetric animals.

By Ingrid Lekk

The left brain leads the right in making neurons

Myriam Roussigne and Steve Wilson
Nodal signalling imposes left-right asymmetry upon neurogenesis in the habenular nuclei

The left and right sides of our brain mediate different cognitive and sensory functions; for instance, the left hemisphere is dominant in processing aspects of language and tool use while the right hemisphere handles visuospatial attention and interpretation of emotion. This feature of the brain is known as functional lateralisation and is thought to improve our cognitive performance as the specialization of each hemisphere broadens the repertoire of tasks that can be undertaken. Brain lateralisation is not unique to humans and underlies asymmetric behaviour in all vertebrates, a common example being visual lateralization, which is manifest as an eye preference that is dependent on what is being looked at.

Functional lateralisation of the brain is a consequence of structural differences between the left and right sides of particular brain regions. One of the best characterised neuroanatomical asymmetries is found in a region of the brain called the epithalamus and in the last ten years, studies of developing zebrafish have led the way in elucidating the mechanisms by which these epithalamic asymmetries develop. The zebrafish epithalamus consists of several groups of neuron (neuronal nuclei) - the pineal complex and a pair of habenular nuclei which display left-right (LR) asymmetries in the proportion of different subtypes of neurons and in their connections (see the publication summary entitled "Brain asymmetry at the level of single cells"). The pineal complex is composed of the symmetrically positioned epiphysis and a left-sided parapineal nucleus that migrates from the midline and connects to the left habenula. The left-sided migration of the parapineal nucleus is dependent upon the activity of a protein called Fgf8 expressed bilaterally in the presumptive habenulae (see the publication summary entitled "Fgf8 signalling breaks symmetry in the brain"). As it migrates, the parapineal sends signals to the left habenula promoting "left-sided" development by these neurons. If the parapineal is removed, the asymmetry between the left and right habenula is considerably reduced.

The sidedness/laterality of parapineal migration and habenulae asymmetries are always concordant (both on the same side) and dependent on the earlier left-sided signalling by Nodal proteins. Importantly, in the absence of one-sided Nodal signalling, neuroanatomical asymmetries develop but with a randomised sidedness: the parapineal migrates to the left or to right side of the epithalamus with an equal probability and the pattern of habenular asymmetry is either normal or mirror reversed correlating with parapineal position. Thus, Nodal signalling is not required for the establishment of structural asymmetry per se but is responsible of the directionality/laterality of asymmetries.

Figure 1: Temporal LR asymmetry in habenular neurogenesis. Images looking down onto the midline of a zebrafish embryo brain. A : cxcr4b expressing neurons appear earlier in the left habenula (arrow) than in the right. The epiphysis is indicated by a dotted blue circle. A’ Staining of all cells in the same brain allows detection of the parapineal (yellow shading) and pineal (blue shading).

The manner in which Nodal signaling imposes left bias in the brain is not known. To address this issue, we explored the involvement of Nodal signalling in promoting the production (neurogenesis) of habenular neurons. We found that a novel marker of habenular precursors/neurons called cxcr4b appears earlier in the left habenula than in the right. This is consistent with previous results from our colleague Hitoshi Okamoto's team showing temporal LR asymmetries in the generation of neuronal sub-types between left and right habenulae. Asymmetry in habenular neurogenesis can be detected before the migration of the parapineal and, indeed, we found that it is independent of the parapineal. In contrast, when we remove the L/R bias in Nodal signaling, habenular neurons appear at the same time in both habenular nuclei. Thus, the activity of Nodal signals in the left epithalamus can directly drive an asymmetry in habenular neurogenesis and this is the first example for a role for Nodal signalling in promoting an asymmetry per se rather than in directing laterality in the brain.

Figure 2. Nodal signalling promotes early asymmetric neurogenesis Images looking down onto the midline of embryonic zebrafish brains showing neurons in green. A and C show normal brains with LR asymmetric neurons (arrows). B and D show brains that don’t have asymmetric Nodal signalling and have LR symmetric neurons (small arrows). The epiphysis is indicated by a dotted blue circle.

The roles/consequences of this Nodal dependent LR asymmetry in habenular neurogenesis are not clear yet but it could be a way by which Nodal biases the orientation of parapineal migration and subsequent habenular asymmetries. This model is consistent with previous data suggesting that the left habenula may provide cues that influence the orientation of parapineal migration. This early asymmetric neurogenesis could also partly influence the later identity of habenular neurons and could consequently be responsible for subtle differences that remain between the left and right habenulae after removal of the parapineal nucleus (see the publication summary entitled "Brain asymmetry at the level of single cells").

How might an early neurogenesis in the left habenula influence the orientation of migration of the parapineal? As we have shown that the protein Fgf8 is required for parapineal migration (see the publication summary entitled "Fgf8 signalling breaks symmetry in the brain"), it will be important to determine if Nodal signalling biases levels of Fgf8 activity, for instance by promoting a L/R asymmetry in the number of fgf8 expressing habenular cells. An alternative possibility is that both asymmetric neurogenesis and directed parapineal migration might be independent consequences of a Nodal-dependent asymmetry in Fgf activity. Our ongoing research is addressing the interactions between Nodal and Fgf signalling pathways in the epithamus.

If you have any further questions, please contact Myriam Roussigne or Steve Wilson

This study was a collaboration with Patrick Blader and our work received financial support from the Wellcome Trust and FEBS.

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Fgf8 signalling breaks symmetry in the brain

Jenny Regan and Steve Wilson

Original paper reference:
An Fgf8-dependent bi-stable cell migratory event establishes CNS asymmetry

Left-right asymmetry is a universal feature of the central nervous system (CNS) and is fundamental to proper brain function. In this study, we sought to answer a question about which virtually nothing was known: "How is symmetry broken in the vertebrate brain?"

The zebrafish brain shows differences between left and right sides in terms of structure, organization and connectivity of nerve cells (neurons). These features have helped to make the zebrafish a focus for studies of brain asymmetry. We have previously shown that the consistent development of brain asymmetries in one direction (laterality or handedness) is dependent on left-sided activity of a Nodal-family signalling protein. Crucially, if Nodal signalling occurs on both sides of the brain or is absent, brain asymmetries still develop, but are randomised, such that normal brain laterality and reversed brain laterality are equally likely outcomes. Therefore, whilst consistent laterality relies on Nodal signalling, development of an asymmetric brain per se does not, and must be dependent on other signals. To uncover the signalling pathways required to break symmetry in the brain, we looked for lines of zebrafish carrying genetic mutations that prevent the development of brain asymmetry.

Images of brains of normal (wild-type, WT left) and ace/fgf8 mutant (right) zebrafish in which all cells are labelled with a red nuclear marker (TOPRO-3) and parapineal neurons with an additional green marker (green fluorescent protein). The parapineal neurons are on the left in the wild-type brain but stuck at the middle in the fgf8 mutant.

We discovered that fish carrying a mutation in the fgf8 gene, called acerebellar (ace), have symmetric brains. Although all relevant brain structures are specified in the mutant, they are unable to develop asymmetrically. This is first evident in the failure of a small group of neural cells, the parapineal, to migrate in a stereotypical leftward arc away from the midline. This leftward migration normally initiates a cascade of events that leads to elaboration of brain asymmetries and culminates in the establishment of asymmetric brain circuits. We found that in ace embryos, the parapineal remains at the midline and never migrates, and later-developing brain structures remain symmetric.

When we looked to see where the fgf8 gene is expressed in normal embryos, surprisingly, we found it on both sides of the brain adjacent to the parapineal, as are some genes turned on in response to Fgf signalling. Supporting the idea that Fgf signals act upon the parapineal, several genes functioning in the Fgf-pathway, including the Fgf-receptor FgfR4, are expressed specifically in this structure.

In order to determine whether signalling by Fgf8 is required for the parapineal nucleus to move leftward from the midline, we provided ace brains with a localised source of Fgf8. This was able to rescue the migration of the parapineal nucleus in ace mutants, however, migration was usually to the left, irrespective of the location of the source of Fgf8. This led us to suspect that another signal acts together with Fgf8 to influence the direction of migration. Indeed, we found that the leftward bias in Fgf-dependent migration is due to left-sided Nodal signalling. In situations where the strong Nodal bias is removed, the Fgf8 source can determine the direction of brain laterality, possibly by acting as an attractant to parapineal cells.

This and other data allowed us to produce a model for generation of brain asymmetry, where left and right sides of the brain compete to attract the parapineal via Fgf8-signalling, initiating a cascade of asymmetric development on the winning side. In normal brains (Panel A below), Nodal signalling strongly biases Fgf-dependent migration to the left. However, Nodal in the absence of Fgf8 is not sufficient to promote migration (Panel C). If Nodal is taken away, the side of the brain that wins the competition is probably that which has stochastically slightly higher levels of Fgf8 (Panel B). Indeed, if we experimentally provide Fgf8 on one side in such situations, that side that wins the competition. The study shows that the combined action of Fgf and Nodal signals ensures the establishment of brain asymmetries with consistent laterality, and suggests that mechanisms to generate asymmetry and direct laterality can be uncoupled and may have evolved sequentially.

Schematic representing Fgf8 and Nodal signalling (top) and resulting brain asymmetry (bottom) in normal (wild-type) embryos (A), in embryos with unbiased Nodal signals (B) and in fgf8 mutants (ace, C). Lh, left habenula; Rh, right habenula. The habenulae are paired structures that produce Fgf8 signals and elaborate asymmetries themselves in response to parapineal migration.

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If you would like to read more about our asymmetry research, please visit our asymmetry research web pages. If you have any more questions about this work, please contact Jenny or Steve Wilson

Our work on this project was primarily funded by the Wellcome Trust

Brain asymmetry at the level of single cells

Isaac Bianco and Stephen Wilson
Original paper reference:
Brain asymmetry is encoded at the level of axon terminal morphology

We use the left and right sides of our brains in different ways and this lateralisation of neural processing is observed throughout the animal kingdom. For instance, in humans, many aspects of the perception and generation of language occur predominantly in the left cerebral hemisphere and in many vertebrate species the left and right visual fields are specialised for processing different types of visual scenes. Presumably, these asymmetries in processing function are associated with left-right differences in the underlying neural architecture wherein the two sides of the brain are specialized for particular computational tasks. Although neuroanatomical asymmetries have been described at the level of differences in overall size, gross connectivity between brain regions and in the types of neurotransmitters used by neurons in equivalent regions on the left and right sides of the brain, very little is known about asymmetries in the microarchitecture of defined neuronal circuits that have distinct information processing roles. One reason for this is that such differences are likely to be relatively subtle. For instance, there could be differences in dendritic and/or axonal arbors - cellular processes by which neurons receive or transmit information to or from other neurons in the circuit, or in the synaptic connections that are formed on the arbors.

In this study, we resolved asymmetry at single-neuron resolution in a structure in the larval zebrafish brain called the habenulo-interpeduncular pathway and identified a previously unrecognized strategy by which neural circuits on the left and right sides of the brain can become distinct. We focused on this particular pathway because asymmetries between the left and right habenular nuclei are amongst the most conserved and conspicuous in the vertebrate brain. Furthermore, we had previously determined that the left and right habenulae establish laterotopic connections with their target, the interpeduncular nucleus (IPN), which lies at the midline of the ventral midbrain: whilst most left habenular axons innervate the dorsal part of the IPN, the majority of right habenular axons connect to the ventral IPN (Aizawa et al, 2005)

In collaboration with Jon Clarke (now at King's College London), we optimised a focal electroporation technique that allows nucleic acids or dyes to be introduced into individual cells in living zebrafish embryos. By expressing a membrane-tethered fluorescent protein, GFP, we were able to visualize the entire morphology of single habenular projection neurons in the intact brain (Fig 1A).

Figure 1 : Two subtypes of habenular projection neuron revealed by focal electroporation. (A) This panel shows an intact fish brain in which the entire morphology of a single right-sided habenular neuron is revealed following electroporation with a construct driving expression of GFP that localizes to the membrane of the neuron. (B and movie 1) An example of a L-typical axonal arbor, formed by most left habenular neurons and only a few right-sided cells. (C and movie 2) An example of a R-typical arbor elaborated by the majority of right habenular neurons. All panels show dorsal views, anterior top Scale bar in (A): 100µm.

Figure 1 : Two subtypes of habenular projection neuron revealed by focal electroporation. (A) This panel shows an intact fish brain in which the entire morphology of a single right-sided habenular neuron is revealed following electroporation with a construct driving expression of GFP that localizes to the membrane of the neuron. (B and movie 1) An example of a L-typical axonal arbor, formed by most left habenular neurons and only a few right-sided cells. (C and movie 2) An example of a R-typical arbor elaborated by the majority of right habenular neurons. All panels show dorsal views, anterior top Scale bar in (A): 100µm.

Movies 1- Movies 2

This led to the identification of two sub-types of habenular projection neuron which have axon terminal arbors with very distinct morphologies. The vast majority of left-sided habenular neurons elaborate axon arbors of one type, which we have called "L-typical" (Fig 1B). These arbors are shaped like a domed crown, localize to the dorsal part of the IPN and extend over a relatively large depth. Such arbors are also formed by a small number of right-sided cells. However, most right habenular neurons instead elaborate "R-typical arbors" which take the form of a flattened spiral and innervate the ventral IPN (Fig 1C). This axon arbor sub-type is also formed by a minority of left habenular cells.

Thus we have identified two neuronal subtypes that display distinct morphologies and connectivity and that are generated on both sides of the brain but in very different numbers. This represents a novel strategy for lateralisation of neuronal circuitry: the same (or very similar) circuit components are generated on both sides of the brain, but differences in the relative ratios of these shared components results in left-right specialization of circuit microarchitecture (Fig 2).

Figure 2: Models for lateralization of neural circuits. (A) In a simple 'scaling' model, equivalent regions on the left and right sides of the brain have the same composition and differ only in overall size. Cortical territories and language-associated fibre tracts appear to show this mode of lateralization. (B) Unique types of neuron or patterns of connectivity might be specified on one side of the brain (indicated by the unique red neurons on the left in this schematic). For example, the parapineal nucleus of neurons is located on the left side of the brain and innervates the left habenula with no equivalent neurons on the right. (C) The same circuitry components are produced on both sides of the brain but in different relative ratios so as to produce unique, lateralized circuits. This is the model we propose for habenulo-interpeduncular circuitry.

How do these different neuronal subtypes contribute to asymmetric processing in the habenulo-interpeduncular pathway? To try to shed light on this question we again turned to focal electroporation and examined the morphology of neurons in the target IPN. We found that whilst some post-synaptic neurons have their dendritic arbors completely confined to either the dorsal or ventral regions of the IPN neuropil (and as such will receive either L-typical or R-typical inputs), others types have dendritic arbors in both sub-regions. This suggests the IPN could integrate inputs from both sides of the brain whilst also containing circuits that preferentially relay left-dominant or right-dominant information to downstream targets.

To understand how asymmetric circuitry develops in the vertebrate brain, we examined the role of the parapineal, a small cluster of neurons that migrates towards and innervates the left habenula and is important for the expression of many of the molecular-genetic asymmetries between the left and right habenulae (see publications page for Concha 2003). In larvae in which the parapineal was removed by laser ablation, both habenulae predominantly innervated the ventral IPN, yielding a connectivity pattern that is superficially 'double right-sided'. However, examination of single neuron morphologies showed that subtle left-rght differences persist in parapineal-ablated embryos, suggesting that other, as yet unidentified developmental signals act to lateralise this highly conserved circuit.

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• This study has been shortlisted for the Biology Prize as part of the annual BioMed Central Research Awards
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If you would like to read more about our asymmetry research, please visit our asymmetry research web pages. If you have any questions about this work, please contact Isaac (i.bianco@ucl.ac.uk) or Steve (s.wilson@ucl.ac.uk).

This project was funded by the Wellcome Trust and an European Communities grant entitled "Evolution and Development of Cognitive, Behavioural and Neural Lateralisation"

From body to brain asymmetry - distinguishing left from right in the developing brain

Matthias Carl and Steve Wilson
Original paper reference:
Wnt/Axin1/β-catenin signaling regulates asymmetric Nodal activation, elaboration, and concordance of CNS asymmetries

The vertebrate brain is an immensely complex structure which exhibits numerous neuroanatomical and functional asymmetries. In humans, some of these asymmetries are evident as biases in hand preference and from the different deficits that arise as a consequence of strokes on left or right sides of the brain. However, it is very difficult to study brain development in humans and so we use animal model systems to study the genetic and cellular mechanisms that underlie brain asymmetry.

Due in part to its small size, relative simplicity and transparency, the zebrafish embryo is particulary well suited to study the genetic pathways that allow the correct establishment of brain asymmetries. In the fish brain, several structures develop prominent asymmetries that are easy to visualise. In the dorsal part of the brain, so called parapineal neurons (nerve cells) are found exclusively on the left side of the brain and also the bilaterally positioned clusters of habenular neurons exhibit asymmetric features (figure 1).

Figure 1. An image of asymmetric neurons in the larval fish brain. The blue neurons are at the midline of the brain, the green parapineal neurons are on the left and the red habenular neurons are differently organised on the left and the right.

In the search for genes that underlie the laterality/handedness of these brain asymmetries, we showed the Nodal signalling pathway plays an important and evolutionary conserved role. Nodal is one of a small number of genetic pathways that allow cells to communicate with each other during embryonic development. Some genes in this signalling cascade are active during a short period of time only on the left side of the brain and determine the subsequent directionality of the migration of parapineal cells from the midline towards the left side of the brain. Subsequent to this, further signals from the parapineal neurons are important to elaborate asymmetries in the habenulae. These various interactions eventually lead to the development of asymmetric neuronal circuits and behaviour.

Figure 2. Images of brains of normal (sibling on the left) and masterblind/Axin1 (mbl on the right) mutant embryos. The habenulae in the top panel are asymmetric in the normal brain but symmetric in the mutant. (Bottom panel) These panels shows the connections made by the left habenula neurons (red) and the right habenula neurons (green). Left and right connections are segregated in the normal embryo but intermixed in the mutant consistent with the brain having symmetric double right character.

Although Nodal signalling in the left brain determines the direction of brain asymmetry, this begs the question of how Nodal signalling is restricted to the left side of the brain in the first place? In our search for genes which influence asymmetric Nodal signalling, we found a zebrafish mutant in which the asymmetry of Nodal is lost and the brains develop symmetrically, as if they are double right-sided (figure 2). These masterblind mutants carry a mutation in Axin1, a gene which acts to turn down the activity of another signalling cascade, the Wnt/β-catenin pathway. We were able to show that Axin1 mediated signaling during very early stages of embryonic development in the future brain is required to establish Nodal gene asymmetry. Too much Wnt/β-catenin signalling at these stages (as happens in the mutant) leads to activation of Nodal signalling on both sides of the brain.

These and other results led us to propose a rather complicated and counter-intuitive model of how Nodal comes to be on the left. A first step is that both sides of the brain are primed to express Nodal but the pathway is switched off on both sides (this step involves the Wnt pathway). Later in development, a signal from the left side of the body signals to the brain to remove the 'off-switch' and consequently Nodal is activated only in the left brain.

So we take a step backwards and now ask why does only the left side of the body send the signal to the brain. It turns out that the Nodal pathway is involved again. It is required to determine the asymmetries in our internal organs like the heart, liver and lungs and in doing this, the pathway appears to have been co-opted to signal left from right in the brain.

Our studies also revealed a later role for Wnt signalling in the communication between the asymmetric brain structures and this is a topic that we are currently following up.

Here's a link to our research description pages.

A related study from Lila Solnica-Krezel's group was published together with ours
Inbal A, Kim SH, Shin J, Solnica-Krezel L. (2007)
Six3 represses nodal activity to establish early brain asymmetry in zebrafish
Neuron 55:407-15

And both studies are reviewed in
Sagasti A (2007)
Three ways to make two sides: genetic models of asymmetric nervous system development
Neuron. 55:345-51

If you have any more questions about this work, please contact Matthias or Steve Wilson

Our work on this project was primarily supported by the BBSRC and Wellcome Trust with some additional funding from the EU.

How are brain asymmetries related to lateralised behaviour?

Anukampa Barth and Steve Wilson
Original paper reference
fsi Zebrafish Show Concordant Reversal of Laterality of Viscera, Neuroanatomy, and a Subset of Behavioral Responses

Although our body looks quite symmetrical on the outside, the localisation of many internal organs such as the heart and liver is asymmetric. Moreover, our brain shows anatomical and functional lateralization. Although this has been known for more than a century, until about 20 years ago our understanding of the differences between the left and right side of the brain was gathered mainly from post-mortem analyses and patients with specific brain lesions (such as strokes affecting one side of the brain). More recently, functional MRI studies in humans and experiments in animal models have helped to reveal more about the relationship between neuroanatomical structures and their function. Not only are our brains asymmetric in terms of structure and activity, some behaviours are also lateralised. An example is handedness in humans, and eye preference for detecting prey or predators in fish and amphibians.

In this publication, we show how we can use zebrafish to study brain asymmetries and how these asymmetries might relate to lateralised behaviours. We made use of a line of fish called frequent-situs-inversus (fsi), in which a proportion of the embryos show a reversal of the asymmetric placement of the viscera. For example, the direction of the looping of the heart is reversed (figure 1A, red arrow), as are the looping of the gut and position of the pancreas. The most conspicuous asymmetries in the CNS are found in the epithalamic region of the dorsal forebrain; in this region there is a left-sided nucleus of neurons called the parapineal and the left and right habenular nuclei show various asymmetries including their connections to other neurons (Figure 1B). Importantly, in fsi embryos with reversed heart looping the brain asymmetries are reversed concordantly (Figure 1).

Figure 1. (A) In normal fish the heart loops from left to right and a small photoreceptive structure in the brain called the parapineal is located to the left side of the midline. Both asymmetries are reversed in the fsi fish line. The pictures show frontal views of young zebrafish embryos. A red arrow indicates the direction of heart looping and the asterisks point to the position of the small parapineal next to the larger pineal nucleus. (B) The left panels show 3-D reconstructions of the fluorescently labeled pineal and parapineal (asterisk) nuclei in normal (top) and reversed fsi fish (bottom). The top right panel depicts the projections of habenular neurons from the left side (red) and right side (green) to a target in the midbrain; in normal fish the left side sends projections to the top (dorsal) part of this target, the right side to the bottom (ventral) part. Again, the laterality of this asymmetry is reversed in fsi fry (bottom right panel)

Some behaviours are lateralised in zebrafish such as eye preference when viewing a conspecific or their own reflection. We wanted to know if this bias would be changed in larval fish (fry) that show a reversal of brain asymmetries. We tested fsi fry that showed a reversal of heart position (which we can see in living fry) and parapineal position (which is visible through expression of a fluorescent transgene in living fry; see Figure 1B left panels). For this test, we place fry into a tank with mirrored walls (Fig 2A; top) and score which eye the fry use to look at their reflection (Fig 2A; bottom). Interestingly, we found that indeed the characteristic eye switching and laterality of eye preference is reversed in fry with reversed CNS asymmetries.

Figure 2. fsi fry with reversed visceral and brain asymmetries show reversal of eye preference when viewing their own reflection. (A) The top panel shows a schematic of the mirror tank used in the test, the bottom panel how we score which eye is used by the fry for viewing. (B) Normal fry (LH, blue line) show a slight bias to use their right eye initially, but then switch to left eye use and back to left eye use. This bias is reversed in fsi fry with reversed asymmetries (RH, pink line).

Using a different test, the swimway, we examined if there is a bias in turning when fry enter a new compartment driven by their preference to move towards the light. Fry are place into a lit start compartment, the light is then switched to the next compartment and the time it takes for the fry to swim into the next compartment (latency) and the laterality of turning is measured. A slight leftward bias was found for the laterality of turning for both groups, normal and reversed (not shown). However, we did see a difference between the groups when we challenged fry by placing a novel object (black stripe) into successive compartments (Fig 3A). Reversed fsi fry appeared to be bolder when exploring spaces featuring novel visual cues; they emerged into successive compartments with less delay (latency) than their normal siblings (Fig 3B).

Figure 3. fsi fry with reversed asymmetries emerge into successive compartments with less delay than their normal siblings when encountering a novel visual object. (A) Fry are placed into the lit start compartment, and are then motivated to enter the next compartment by turning off the light in the start compartment and turning it on in the adjacent compartment. This is repeated for 3 successive compartments, each successive compartments feature a novel object (black stripe) on the side wall. (B) Fry are encountering a novel visual object upon emerging into the new compartment. Numbers 1, 2 and 3 on the horizontal axis show the number of consecutive compartments entered; the vertical axis shows the average delay of entering the next compartment after switching the light (in seconds).

Our findings support the idea that asymmetries in the brain are directly correlated to at least some lateralised behaviours. In addition, we found that fsi fry with reversed CNS asymmetries show a distinct behaviour compared to their normal siblings; they are less afraid to move into novel spaces, even if they encounter novel visual stimuli. This suggests that some lateralised behaviour is reversed in fry with reversed brain asymmetries and that brain reversal can lead to novel behaviours such as increased boldness. We are currently expanding these studies to include a wider range of behavioural tests.

Further information
McManus C. (2005) Reversed bodies, reversed brains, and (some) reversed behaviors: of zebrafish and men. Dev Cell. 8:796-7
Lin SY, Burdine RD (2005) Brain asymmetry: switching from left to right. Curr Biol. 15:R343-5

If you have questions about this work please visit our asymmetry research pages or contact Anukampa Barth or Steve Wilson

This study was a collaboration with Richard Andrew's group at the University of Sussex. Work from our lab on this study was supported by the Wellcome Trust.