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Leaning to the left: laterality in the zebrafish forebrain Marnie E. Halpern1, Jennifer O. Liang1,2 and Joshua T. Gamse1 1 2
Carnegie Institution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, MD 21210, USA Case Western Reserve University, Biology Department, 10900 Euclid Avenue, Cleveland, OH 44106, USA
How the brain becomes lateralized is poorly understood. By contrast, much is known about molecular cues that specify the left –right axis of the body, fashioning the asymmetric morphology and positioning of the visceral organs. In zebrafish, the Nodal signaling pathway functions in visceral asymmetry and also in the embryonic brain, to bias laterality of the epithalamus. Formation of an asymmetric pineal complex differentially influences adjacent diencephalic nuclei, the left and right habenulae, which acquire distinctive molecular and cellular features. Results from the genetically tractable zebrafish system provide a promising entry point for exploring how left–right biases are established and propagated in the developing vertebrate brain. Anatomical differences between the left and right hemispheres have been implicated in many aspects of brain function and dysfunction, including the developmental disorders of schizophrenia, depression, autism and dyslexia [1– 7]. Despite potential medical relevance, we have a limited understanding of the origin of morphological asymmetry in the brain and its importance in predominantly lateralized cognitive processes such as language generation or perception. Hemispheric specialization has long been recognized: the ancient Greeks knew that injuries confined to one side of the head produce different symptoms from those sustained on the opposite side [8]. In the 19th century, studies of patients with selective language deficits after stroke or other localized assault allowed cognitive tasks to be assigned to discrete brain regions [9]. A resurgence of interest in hemispheric asymmetry followed Geschwind and Levitsky’s 1968 report, in which precise measurement of human brain sections confirmed that the left and right cortex possess distinct anatomical features [10]. The planum temporale, a region of the posterior temporal lobe, is significantly larger on the left in most individuals. The length and slope of the Sylvian fissure, a deep cleft in this region, also differs between hemispheres, and the right frontal lobe protrudes more anteriorly than the left [11 – 14]. Modern imaging concurs with these findings [14] and reveals comparable left– right differences in the brains of higher primates [15,16]. Surprisingly little work in model systems has addressed how anatomical asymmetry arises in the vertebrate brain. Corresponding author: Marnie E. Halpern ([email protected]).
To some extent, this is owing to the subtle nature of left – right differences in the largely symmetrical brain and to the lack of molecular tools. The value of comparative studies was also in doubt because of the long-held view that laterality is unique to the human neocortex. The discovery in the 1970s that left– right paired nerves innervating the avian syrinx have differential activities in the control of song production challenged this view [17]. Asymmetric behaviors exhibited by fish, birds, amphibians and rodents have since provided a strong argument for functional lateralization being a universal and evolutionarily ancient property of the vertebrate brain [18 –23]. In only a few cases, however, such as the elegant studies of chicken and pigeon visual systems [23], have behavioral biases been directly linked with neuroanatomical asymmetry. Molecular and genetic approaches that provided insight into asymmetric development of the viscera [24,25] are also proving valuable for exploring left– right differences in the zebrafish nervous system. This review summarizes recent work on the dorsal diencephalic region of the forebrain, the epithalamus (Fig. 1), which in zebrafish, as in many lower vertebrates, is organized asymmetrically. Analyses of zebrafish mutants indicate that the Nodal signaling cassette mediating visceral asymmetry also controls anatomical laterality of the epithalamus. An unexpected finding is that lateralization appears sequentially, with an asymmetry in one region, the pineal complex, influencing left– right identity of neighboring diencephalic nuclei. Laterality of the epithalamus Left– right differences in epithalamic anatomy have been documented for 150 years [26,27] and have been described for a wide range of species from primitive fish to mammals, as comprehensively reviewed by Concha and Wilson [28]. The epithalamus contains the pineal complex and the bilaterally paired dorsal habenular nuclei connected by a prominent commissure. The pineal complex often consists of two components: the medial pineal organ (or epiphysis) and an associated, more variable structure referred to as the frontal organ (amphibians), parietal organ (reptiles) or parapineal organ (fish) [29]. Although sidedness differs between species, the parapineal is asymmetrically positioned in the fish diencephalon [30– 33]. The habenular nuclei can appear relatively symmetrical or differ greatly. Especially noteworthy is the work of Kemali [34], who found a striking difference in the habenulae of the frog
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Fig. 1. Zebrafish epithalamic development. (a) Schematic side view of a two-dayold zebrafish larval head, showing the pineal domain (P, blue) in the dorsal diencephalon (Di). (b) In a four-day-old larva (dorsal view), the pineal complex consists of the pineal anlage (P) and the left-sided parapineal (Pp). The adjacent diencephalic nuclei, the left and right dorsal habenulae (LH and RH, respectively; red), are connected by a prominent commissure and send efferents through the fasciculus retroflexus (FR) to the midbrain. (c) In the adult epithalamus (dorsal view), the photoreceptive pineal end-vesicle (P) is connected to a stalk emerging from the diencephalic roof; the small parapineal remains closely apposed to the left habenula. Abbreviations: Ot, optic tectum; Te, telencephalon.
Rana esculenta, with the left habenula being larger and consisting of more compartments than the right. Numerous morphological, biochemical and physiological left– right habenular differences have been discovered in a variety of vertebrates [27,28,35–39]. The pineal is known to function in melatonin synthesis and regulation of circadian activity but the role of the parapineal is less clear (although the photoreceptive parietal organ of lizards is structurally analogous to the eye [29]). The dorsal habenulae serve as a relay in an evolutionarily conserved conduction system that conveys information from telencephalic nuclei to the midbrain via the fasciculus retroflexus (also known as the habenulointerpeduncular tract) and that integrates neural activity with the autonomic and endocrine systems [40]. Functional dissection has been challenging because the habenular system mediates behaviors as diverse as olfactory perception, mating, feeding, hormonal release, the response to aversive stimuli, and reward [40,41]. Intriguingly, the size asymmetry of the habenular nuclei is sexually dimorphic and coupled to testosterone levels in chickens [42,43] and shows seasonal variation in Rana esculenta [44]. Molecular and structural asymmetry in the zebrafish diencephalon Attention refocused on epithalamic asymmetry with the discovery that components of the Nodal pathway are expressed on the left side of the zebrafish diencephalon briefly during embryogenesis [45 – 51]. The transforminggrowth-factor b (TGF-b) family member Nodal was initially identified in the mouse as an essential signal in mesoderm formation at gastrulation [52,53] and as a determinant of the left– right body axis [54]. Left-sided http://tins.trends.com
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expression of nodal-related genes in the lateral-plate mesoderm is conserved in all chordates, as is expression of the Nodal-regulated gene pitx2, which encodes a homeodomain transcription factor [24,25,55]. Careful double labeling demonstrated that genes encoding the Nodal-related factor Cyclops (Cyc/Ndr2), Pitx2 and the Nodal antagonist Antivin/Lefty 1 (Lft1) are all transiently expressed on the left side of the bilateral pineal anlage [49] (Fig. 2). The genes one-eyed pinhead (oep), which encodes an essential cofactor for Nodal signaling [56], and schmalspur (sur), which encodes a FoxH1/Fast-1 transcription factor that is active in Nodal receptive cells [57,58], are transcribed in a partially overlapping domain. Thus, the left diencephalon of the zebrafish embryo possesses the same intercellular signaling cassette as the left lateral-plate mesoderm and appears competent both to produce and to receive Nodal signals. Additional anatomical and molecular asymmetries appear as the larval epithalamus develops (Fig. 2). Serotonin N-acetyltransferase expression provided the first evidence of distinct pineal and left-sided parapineal cellular clusters [59], which are also immunoreactive for the transcription factor Islet1 and the photopigment component Opsin [60]. The parapineal forms several hours after nodal expression disappears from the left pineal anlage, as revealed by expression of otx5, an early parapineal marker [61,62]. Fate-mapping and cell ablation indicate that the parapineal is derived from the same embryonic anlage that produces the pineal (J.T. Gamse, K. Cygnar and M.E. Halpern, unpublished). While the pineal later evaginates from the diencephalic roof to produce the elongated stalk and photoreceptive endvesicle, the parapineal remains a rudimentary structure, persisting near the base of the pineal stalk closely apposed to the left habenula [62]. Size differences between the habenulae are less prominent in zebrafish than in primitive fish or Rana, with the left habenula of 4-day-old zebrafish larvae being , 15 – 20% larger than that on the right [62]. More impressive are the dense neuropil of the left habenula and expanded expression of the recently identified leftover (lov) gene [60,62]. Zebrafish lov encodes an unknown protein that contains an oligomerization domain similar to Shaker-related Kþ channel subunits [63]. lov is transcribed throughout the left habenula at high levels, but at lower levels and in a limited region on the right. Expression is asymmetric from its onset and persists in this pattern throughout life [62]. Left-sided expression of the Nodal pathway in the embryonic pineal, the left-positioned parapineal, and the enlarged neuropil and lov expression in the left habenula are found in the vast majority of zebrafish, indicating a strong population-related bias to maintain directional asymmetry of the epithalamus. Global regulation of left– right asymmetry Lessons learned from studies of visceral asymmetry are useful for understanding how left –right differences might be set up in the brain. Many mutations in mouse and zebrafish are known to cause left–right randomization (antisymmetry) in which, for example, the normally rightward
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Fig. 2. Sequential appearance of epithalamic asymmetry. (a) In the segmenting zebrafish embryo (20 hours post-fertilization), cyclops/nodal-related 2 (cyc/ndr2, blue), antivin/lefty (lft1) and pitx2 (not shown) are briefly transcribed on the left side of the presumptive pineal, which is defined by its bilateral expression of the floating head gene (red) [79] (i). By two days post-fertilization, the parapineal (arrowhead) has formed to the left of the pineal, as revealed by otx5 expression (ii). In four-day-old larvae, the dorsal habenular nuclei express the leftover gene (iii) and exhibit differences in neuropil density (iv) in a characteristic left –right pattern. (b,c) Schematic of left– right differences in the epithalamic development of wild-type (b) and mutant (c) zebrafish. Mutations that perturb midline formation or Nodal signaling result in bilateral or absent pineal gene expression, respectively. In both classes of mutants, parapineal sidedness and habenular laterality are left –right randomized, but always in a concordant manner. All panels show dorsal views.
looping of the heart tube is reversed in half of affected individuals [24,25]. In zebrafish, defects in visceral asymmetry result from a variety of perturbations, including defective tissue patterning at gastrulation, abnormal midline formation and loss of Nodal pathway components [45–51]. The same early-acting mechanisms also regulate epithalamic asymmetry. However, some zebrafish mutants show discordance in the direction of visceral and brain laterality [45,49–51], demonstrating that there are both global and tissue-specific aspects to left–right determination. With respect to global left– right specification, it is still unresolved what the first symmetry-breaking event is and whether it occurs and is propagated similarly in all vertebrates. Recent work in Xenopus implicates asymmetric Hþ and Kþ fluxes and directional gap-junction communication as very early components of left– right patterning that lead to asymmetric nodal expression in the lateral-plate mesoderm [64]. Genetic and cellular studies in mice support a monocilia-mediated mechanism that initiates molecular left– right asymmetry through the directional movement or perception of signals. The socalled nodal flow generated by monociliated cells in the embryonic node is disrupted in mouse mutants showing left –right defects [24]. Although epithelial cells lining Kupffer’s vesicle in the zebrafish also possess motile cilia [65], this structure probably develops too late to qualify as the initiator of left– right axis formation. Sequential development of epithalamic laterality Epithalamic asymmetry arises sequentially in zebrafish and appears to be intimately connected with morphogenesis of the pineal complex. Some insight into this process came from mutants that have disrupted midline formation and http://tins.trends.com
that later express cyc, lft1 and pitx2 on both sides of the pineal anlage [45 –51]. Their parapineal develops either to the left or right of the pineal and is only rarely (, 1%) duplicated bilaterally. In half of such mutant larvae, the neuropil and lov habenular asymmetries are left –right reversed in parallel [60,62]. By contrast, mutations that inactivate Nodal signaling or reception inhibit left-sided gene expression in the pineal anlage [60,62]. This finding allows the function of the Nodal diencephalic asymmetry to be assessed. For example, mutants lacking the cofactor One-eyed pinhead can be rescued past the requirement for Nodal signaling at gastrulation by injection of oep RNA at the one-cell stage [56]. Injected embryos later lack left-sided gene expression in the lateral-plate mesoderm and pineal anlage, yet they develop into adult fish [60,62,66]. In the brains of rescued oep adults, the pineal stalk frequently emerges from the diencephalic roof at an inappropriate position relative to the usual location just left of the midline (Fig. 3). Asymmetric Nodal signaling is not required for morphogenesis of the pineal organ but, rather, it positions outgrowth of the anlage along the left– right axis of the diencephalon [49]. In addition, Nodal signaling influences parapineal position and habenular laterality [60 – 62]. As in midline mutants, half of rescued oep larvae display a right-sided parapineal and left – right reversal of habenular asymmetry (Fig. 2). Therefore, bilateral activation and absence of Nodal activity in the pineal anlage have the same consequence, randomizing the directionality of epithalamic asymmetry across the population. Regardless of sidedness, laterality of the parapineal and habenulae always correspond, suggesting that they are
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Fig. 3. Left –right displacement of the pineal stalk. (a) In adult zebrafish, the pineal stalk usually emanates from the dorsal diencephalic roof with a slight left bias. (b) The left– right position is frequently shifted in mutants that lacked Nodal signaling in their embryonic pineal anlage. (c) Summary diagram showing the midpoint of the pineal stalk relative to the midline of the adult brain. Rescued one-eyed pinhead (oep) mutants (pink circles) show a greater variation in pineal organ placement along the left– right axis of the diencephalon than wild-type zebrafish (blue circles).
coordinately regulated [60,62]. Alternatively, the parapineal itself could modulate left– right identity of the adjacent habenula. In fact, axonal tracing studies demonstrate that in some species the parapineal preferentially innervates the left habenula [28,67]. Strong support for a direct role for the parapineal in habenular laterality comes from laser ablation experiments (Fig. 4). After selective destruction of the parapineal, both habenulae develop but the left habenula is indistinguishable from the right. Conversely, in rare cases where parapineal organs are duplicated to the left and right of the pineal, both habenulae adopt the denser neuropil and extensive lov expression characteristic of the left habenula (i.e. there is left isomerization) [62]. The zebrafish studies promote a model in which global signals set up a left –right bias in cyc, lft1 and pitx2 transcription in the embryonic diencephalon, as they do in the left lateral-plate mesoderm. Asymmetric activation of flh:GFP
the Nodal signaling pathway regulates laterality of the pineal complex, normally directing the parapineal to form to the left of the pineal anlage. The left-sided parapineal promotes the differentiation of left-specific properties in the adjacent brain nucleus, an interaction that potentially could persist in the adult brain. An alternative interpretation, which has not been ruled out, is that the parapineal serves to reinforce or maintain a predetermined laterality in the habenular region. In either case, the nature of the interaction between the parapineal and left habenula is unknown, although close proximity between the two structures appears to be necessary. The strength of the zebrafish system is its amenability to mutagenesis and to genetic dissection of developmental processes [68]. As first demonstrated for hindbrain segmentation [69], mutational screening of the basis of gene expression patterns is readily performed, yielding specific mutations that cause subtle alterations that are not detected in morphology screens. Such an approach using lov should identify early-acting genes that regulate laterality of the brain and viscera as well as those controlling brain-specific events. Significance of epithalamic asymmetry Countless questions remain concerning the importance and universality of diencephalic asymmetry. Laterality of the zebrafish epithalamus is robustly preserved in individuals and at the population level (99% of larvae develop epithalamic asymmetry and 95% have the leftbiased pattern), implying that directional asymmetry confers an advantage for the species. Left– right preferences in eye usage [70– 73] and directional biases in swimming in some teleost fish [74] are thought to mediate social behaviors such as schooling, feeding and the escape response. Whether diencephalic asymmetry is required for expression of lateralized larval and adult behaviors is presently unknown; however, mutant strains should permit rigorous testing of the correlation between anatomy and function. Left– right differences in the habenulae are likely to have an impact on other brain regions. As reported for some fish and frogs, left –right habenular efferents of the fasciculus retroflexus could vary [35,67],
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Fig. 4. The parapineal mediates habenular asymmetry. The pineal and parapineal can be visualized in vivo in floating head: green fluorescent protein ( flh:GFP) transgenic embryos [62], allowing selective laser-mediated cell ablation (red cross). Ablation of cells to the right of the pineal complex (control ablated) does not affect the later expression of symmetric (cpd2, gray) or asymmetric (lov, red) habenular markers, or expression of otx5 in the pineal complex (blue). By contrast, the expanded lov expression pattern and denser neuropil (not shown) characteristic of the left habenula do not appear following destruction of the parapineal (confirmed by loss of the otx5 parapineal domain). In its size and molecular properties, the left habenula resembles the right in parapineal-ablated larvae. http://tins.trends.com
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leading to differential synaptic activity at the target interpeduncular nucleus. Telencephalic inputs might differ if left– right habenulae express different molecular cues when connections are established. Zebrafish transgenic lines should allow direct visualization of the habenular circuitry through spatially regulated expression of green fluorescent protein (GFP) variants. A pressing issue is whether the zebrafish studies will reveal general strategies for constructing brain asymmetry and, thus, provide clues about the evolutionary events that set the framework for the sophisticated hemispheric specialization of the human cortex. Anatomical asymmetry of the diencephalon is an ancient property of the vertebrate lineage that seems more prominent in evolutionarily lower taxa [28], perhaps reflecting a diminished need for its preservation as laterality became fixed and complexity increased in other forebrain regions. Neuroanatomical asymmetry is also found outside the cortex, in thalamic nuclei, the basal ganglia and aspects of the neuroendocrine system [75– 77]. An added complication is that asymmetric morphologies are not exclusively under genetic control but are also the product of hormonal regulation, environmental conditions and experience [23]. Despite intensive searching, there is no evidence yet that the Nodal pathway functions unilaterally in the embryonic brain of mouse, chick or Xenopus, even though Nodal activation in the left lateral-plate mesoderm is conserved. In mammals, molecular left –right differences might be transient or confined to small groups of neurons and, thus, challenging to uncover. The identification of genes such as lov, which is expressed in the zebrafish habenulae throughout life and belongs to a vertebrate multi-gene family [62], could be an asset for comparative studies. Whole genome approaches, such as microarray technology, that utilize probes derived from left-sided versus right-sided brain tissue might prove sensitive enough to detect other differentially expressed genes [78]. From studies of the photoreceptive pineal complex and habenulae, Kemali proposed that left– right asymmetry was ‘imposed onto the brain from the periphery’ and contemplated whether lateralized brain functions had their evolutionary origin in the anatomical asymmetry of the epithalamus [27]. Genetic screens in model systems and the application of molecular methods to diverse species provide a unique opportunity to explore the development, conservation and function of diencephalic asymmetry. Acknowledgements We are grateful to Christine Thisse and Bernard Thisse for generously providing zebrafish brain markers and to Chris Wright and Luis Puelles for valuable discussions. Described research was supported in part by the NSF (M.E.H.) and by post-doctoral fellowships from NRSA (J.O.L.) and the American Cancer Society (J.T.G.).
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35 Kemali, M. and Guglielmotti, V. (1977) An electron microscope observation of the right and the two left portions of the habenular nuclei of the frog. J. Comp. Neurol. 176, 133 – 148 36 Kemali, M. and Guglielmotti, V. (1982) Quantitative evaluation of the number of fibers in the right and left fasciculus retroflexus using horseradish peroxidase. Z. Mikrosk. Anat. Forsch. 96, 750 – 754 37 Morgan, M.J. et al. (1973) Development of left – right asymmetry in the habenular nuclei of Rana temporaria. J. Comp. Neurol. 149, 203– 214 38 Guglielmotti, V. and Fiorino, L. (1999) Nitric oxide synthase activity reveals an asymmetrical organization of the frog habenulae during development: a histochemical and cytoarchitectonic study from tadpoles to the mature Rana esculenta, with notes on the pineal complex. J. Comp. Neurol. 411, 441– 454 39 Wehrmaker, A. (1969) Right – left asymmetry and situs inversus in Triturus alpestris. Wilhelm Roux’s Arch. Dev. Biol. 163, 1 – 32 40 Sutherland, R.J. (1982) The dorsal diencephalic conduction system: a review of the anatomy and functions of the habenular complex. Neurosci. Biobehav. Rev. 6, 1 – 13 41 Sandyk, R. (1991) Relevance of the habenular complex to neuropsychiatry: a review and hypothesis. Int. J. Neurosci. 61, 189 – 219 42 Gurusinghe, C.J. and Ehrlich, D. (1985) Sex-dependent structural asymmetry of the medial habenular nucleus of the chicken brain. Cell Tissue Res. 240, 149 – 152 43 Gurusinghe, C.J. et al. (1986) The influence of testosterone on the sexdependent structural asymmetry of the medial habenular nucleus of the chicken. J. Comp. Neurol. 253, 153– 162 44 Kemali, M. et al. (1990) The asymmetry of the habenular nuclei of female and male frogs in spring and winter. Brain Res. 517, 251– 255 45 Sampath, K. et al. (1998) Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling. Nature 395, 185 – 189 46 Rebagliati, M.R. et al. (1998) Zebrafish nodal-related genes are implicated in axial patterning and establishing left – right asymmetry. Dev. Biol. 199, 261 – 272 47 Rebagliati, M.R. et al. (1998) cyclops encodes a nodal-related factor involved in midline signaling. Proc. Natl. Acad. Sci. U. S. A. 95, 9932 – 9937 48 Thisse, C. and Thisse, B. (1999) Antivin, a novel and divergent member of the TGFb superfamily, negatively regulates mesoderm induction. Development 126, 229 – 240 49 Liang, J.O. et al. (2000) Asymmetric nodal signaling in the zebrafish diencephalon positions the pineal organ. Development 127, 5101– 5112 50 Essner, J.J. et al. (2000) Mesendoderm and left – right brain, heart and gut development are differentially regulated by pitx2 isoforms. Development 127, 1081– 1093 51 Bisgrove, B.W. et al. (2000) Multiple pathways in the midline regulate concordant brain, heart and gut left – right asymmetry. Development 127, 3567 – 3579 52 Conlon, F.L. et al. (1994) A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. Development 120, 1919– 1928 53 Zhou, X. et al. (1993) Nodal is a novel TGF-b-like gene expressed in the mouse node during gastrulation. Nature 361, 543 – 547 54 Collignon, J. et al. (1996) Relationship between asymmetric nodal expression and the direction of embryonic turning. Nature 381, 155 – 158 55 Boorman, C. and Shimeld, S.M. (2002) The evolution of left – right asymmetry of chordates. BioEssays 24, 1004– 1011
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56 Gritsman, K. et al. (1999) The EGF-CFC protein One-eyed pinhead is essential for nodal signaling. Cell 97, 121– 132 57 Pogoda, H.M. et al. (2000) The zebrafish forkhead transcription factor FoxH1/Fast1 is a modulator of nodal signaling required for organizer formation. Curr. Biol. 10, 1041 – 1049 58 Sirotkin, H.I. et al. (2000) Fast1 is required for the development of dorsal axial structures in zebrafish. Curr. Biol. 10, 1051 – 1054 59 Gothilf, Y. et al. (1999) Zebrafish serotonin N-acetyltransferase-2: marker for development of pineal photoreceptors and circadian clock function. Endocrinology 140, 4895– 4903 60 Concha, M.L. et al. (2000) A nodal signaling pathway regulates the laterality of neuroanatomical asymmetries in the zebrafish forebrain. Neuron 28, 399 – 409 61 Gamse, J.T. et al. (2002) Otx5 regulates genes that show circadian expression in the zebrafish pineal complex. Nat. Genet. 30, 117 – 121 62 Gamse, J.T. et al. (2003) The parapineal mediates left–right asymmetry in the zebrafish diencephalon. Development 130, 1059–1068 63 Papazian, D.M. (1999) Potassium channels: some assembly required. Neuron 23, 7 – 10 64 Levin, M. et al. (2002) Asymmetries in Hþ/Kþ-ATPase and cell membrane potentials comprise a very early step in left – right patterning. Cell 111, 77 – 89 65 Essner, J.J. et al. (2002) Conserved function for embryonic nodal cilia. Nature 418, 37 – 38 66 Yan, Y.T. et al. (1999) Conserved requirement for EGF-CFC genes in vertebrate left – right axis formation. Genes Dev. 13, 2527 – 2537 67 Braford, M.R. and Northcutt, R.G. (1983) Organization of the Diencephalons and Pretectum of the Ray-Finned Fishes (Vol. 2), University of Michigan Press 68 Haffter, P. et al. (1996) The identification of genes with unique and essential functions in the development of the zebrafish Danio rerio. Development 123, 1 – 36 69 Moens, C.B. and Fritz, A. (1999) Techniques in neural development. Methods Cell Biol. 59, 253 – 272 70 Miklosi, A. and Andrew, R.J. (1999) Right eye use associated with decision to bite in zebrafish. Behav. Brain Res. 105, 199 – 205 71 Miklosi, A. et al. (2001) Role of right hemifield in visual control of approach to target in zebrafish. Behav. Brain Res. 122, 57 – 65 72 Sovrano, V.A. et al. (2001) Lateralization of response to social stimuli in fishes: a comparison between different methods and species. Physiol. Behav. 74, 237 – 244 73 Bisazza, A. et al. (2000) Population lateralisation and social behaviour: a study with 16 species of fish. Laterality 5, 269 – 284 74 Heuts, B.A. (1999) Lateralization of trunk muscle volume, and lateralization of swimming turns of fish responding to external stimuli. Behav. Processes 47, 113 – 124 75 Koshiba, M. et al. (2003) Light-dependent development of asymmetry in the ipsilateral and contralateral thalamofugal visual projections of the chick. Neurosci. Lett. 336, 81 – 84 76 Gerendai, I. and Hala´sz, B. (1997) Neuroendocrine asymmetry. Front. Neuroendocrinol. 18, 354 – 381 77 Ifthikharuddin, S.F. et al. (2000) MR volumetric analysis of the human basal ganglia: normative data. Acad. Radiol. 7, 627 – 634 78 Geschwind, D.H. et al. (2001) Molecular approaches to cerebral laterality: development and neurodegeneration. Am. J. Med. Genet. 101, 370 – 381 79 Masai, I. et al. (1997) floating head and masterblind regulate neuronal patterning in the roof of the forebrain. Neuron 18, 43 – 57
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Leaning to the left: laterality in the zebrafish forebrain Marnie E. Halpern1, Jennifer O. Liang1,2 and Joshua T. Gamse1 1 2
Carnegie Institution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, MD 21210, USA Case Western Reserve University, Biology Department, 10900 Euclid Avenue, Cleveland, OH 44106, USA
How the brain becomes lateralized is poorly understood. By contrast, much is known about molecular cues that specify the left –right axis of the body, fashioning the asymmetric morphology and positioning of the visceral organs. In zebrafish, the Nodal signaling pathway functions in visceral asymmetry and also in the embryonic brain, to bias laterality of the epithalamus. Formation of an asymmetric pineal complex differentially influences adjacent diencephalic nuclei, the left and right habenulae, which acquire distinctive molecular and cellular features. Results from the genetically tractable zebrafish system provide a promising entry point for exploring how left–right biases are established and propagated in the developing vertebrate brain. Anatomical differences between the left and right hemispheres have been implicated in many aspects of brain function and dysfunction, including the developmental disorders of schizophrenia, depression, autism and dyslexia [1– 7]. Despite potential medical relevance, we have a limited understanding of the origin of morphological asymmetry in the brain and its importance in predominantly lateralized cognitive processes such as language generation or perception. Hemispheric specialization has long been recognized: the ancient Greeks knew that injuries confined to one side of the head produce different symptoms from those sustained on the opposite side [8]. In the 19th century, studies of patients with selective language deficits after stroke or other localized assault allowed cognitive tasks to be assigned to discrete brain regions [9]. A resurgence of interest in hemispheric asymmetry followed Geschwind and Levitsky’s 1968 report, in which precise measurement of human brain sections confirmed that the left and right cortex possess distinct anatomical features [10]. The planum temporale, a region of the posterior temporal lobe, is significantly larger on the left in most individuals. The length and slope of the Sylvian fissure, a deep cleft in this region, also differs between hemispheres, and the right frontal lobe protrudes more anteriorly than the left [11 – 14]. Modern imaging concurs with these findings [14] and reveals comparable left– right differences in the brains of higher primates [15,16]. Surprisingly little work in model systems has addressed how anatomical asymmetry arises in the vertebrate brain. Corresponding author: Marnie E. Halpern ([email protected]).
To some extent, this is owing to the subtle nature of left – right differences in the largely symmetrical brain and to the lack of molecular tools. The value of comparative studies was also in doubt because of the long-held view that laterality is unique to the human neocortex. The discovery in the 1970s that left– right paired nerves innervating the avian syrinx have differential activities in the control of song production challenged this view [17]. Asymmetric behaviors exhibited by fish, birds, amphibians and rodents have since provided a strong argument for functional lateralization being a universal and evolutionarily ancient property of the vertebrate brain [18 –23]. In only a few cases, however, such as the elegant studies of chicken and pigeon visual systems [23], have behavioral biases been directly linked with neuroanatomical asymmetry. Molecular and genetic approaches that provided insight into asymmetric development of the viscera [24,25] are also proving valuable for exploring left– right differences in the zebrafish nervous system. This review summarizes recent work on the dorsal diencephalic region of the forebrain, the epithalamus (Fig. 1), which in zebrafish, as in many lower vertebrates, is organized asymmetrically. Analyses of zebrafish mutants indicate that the Nodal signaling cassette mediating visceral asymmetry also controls anatomical laterality of the epithalamus. An unexpected finding is that lateralization appears sequentially, with an asymmetry in one region, the pineal complex, influencing left– right identity of neighboring diencephalic nuclei. Laterality of the epithalamus Left– right differences in epithalamic anatomy have been documented for 150 years [26,27] and have been described for a wide range of species from primitive fish to mammals, as comprehensively reviewed by Concha and Wilson [28]. The epithalamus contains the pineal complex and the bilaterally paired dorsal habenular nuclei connected by a prominent commissure. The pineal complex often consists of two components: the medial pineal organ (or epiphysis) and an associated, more variable structure referred to as the frontal organ (amphibians), parietal organ (reptiles) or parapineal organ (fish) [29]. Although sidedness differs between species, the parapineal is asymmetrically positioned in the fish diencephalon [30– 33]. The habenular nuclei can appear relatively symmetrical or differ greatly. Especially noteworthy is the work of Kemali [34], who found a striking difference in the habenulae of the frog
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Fig. 1. Zebrafish epithalamic development. (a) Schematic side view of a two-dayold zebrafish larval head, showing the pineal domain (P, blue) in the dorsal diencephalon (Di). (b) In a four-day-old larva (dorsal view), the pineal complex consists of the pineal anlage (P) and the left-sided parapineal (Pp). The adjacent diencephalic nuclei, the left and right dorsal habenulae (LH and RH, respectively; red), are connected by a prominent commissure and send efferents through the fasciculus retroflexus (FR) to the midbrain. (c) In the adult epithalamus (dorsal view), the photoreceptive pineal end-vesicle (P) is connected to a stalk emerging from the diencephalic roof; the small parapineal remains closely apposed to the left habenula. Abbreviations: Ot, optic tectum; Te, telencephalon.
Rana esculenta, with the left habenula being larger and consisting of more compartments than the right. Numerous morphological, biochemical and physiological left– right habenular differences have been discovered in a variety of vertebrates [27,28,35–39]. The pineal is known to function in melatonin synthesis and regulation of circadian activity but the role of the parapineal is less clear (although the photoreceptive parietal organ of lizards is structurally analogous to the eye [29]). The dorsal habenulae serve as a relay in an evolutionarily conserved conduction system that conveys information from telencephalic nuclei to the midbrain via the fasciculus retroflexus (also known as the habenulointerpeduncular tract) and that integrates neural activity with the autonomic and endocrine systems [40]. Functional dissection has been challenging because the habenular system mediates behaviors as diverse as olfactory perception, mating, feeding, hormonal release, the response to aversive stimuli, and reward [40,41]. Intriguingly, the size asymmetry of the habenular nuclei is sexually dimorphic and coupled to testosterone levels in chickens [42,43] and shows seasonal variation in Rana esculenta [44]. Molecular and structural asymmetry in the zebrafish diencephalon Attention refocused on epithalamic asymmetry with the discovery that components of the Nodal pathway are expressed on the left side of the zebrafish diencephalon briefly during embryogenesis [45 – 51]. The transforminggrowth-factor b (TGF-b) family member Nodal was initially identified in the mouse as an essential signal in mesoderm formation at gastrulation [52,53] and as a determinant of the left– right body axis [54]. Left-sided http://tins.trends.com
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expression of nodal-related genes in the lateral-plate mesoderm is conserved in all chordates, as is expression of the Nodal-regulated gene pitx2, which encodes a homeodomain transcription factor [24,25,55]. Careful double labeling demonstrated that genes encoding the Nodal-related factor Cyclops (Cyc/Ndr2), Pitx2 and the Nodal antagonist Antivin/Lefty 1 (Lft1) are all transiently expressed on the left side of the bilateral pineal anlage [49] (Fig. 2). The genes one-eyed pinhead (oep), which encodes an essential cofactor for Nodal signaling [56], and schmalspur (sur), which encodes a FoxH1/Fast-1 transcription factor that is active in Nodal receptive cells [57,58], are transcribed in a partially overlapping domain. Thus, the left diencephalon of the zebrafish embryo possesses the same intercellular signaling cassette as the left lateral-plate mesoderm and appears competent both to produce and to receive Nodal signals. Additional anatomical and molecular asymmetries appear as the larval epithalamus develops (Fig. 2). Serotonin N-acetyltransferase expression provided the first evidence of distinct pineal and left-sided parapineal cellular clusters [59], which are also immunoreactive for the transcription factor Islet1 and the photopigment component Opsin [60]. The parapineal forms several hours after nodal expression disappears from the left pineal anlage, as revealed by expression of otx5, an early parapineal marker [61,62]. Fate-mapping and cell ablation indicate that the parapineal is derived from the same embryonic anlage that produces the pineal (J.T. Gamse, K. Cygnar and M.E. Halpern, unpublished). While the pineal later evaginates from the diencephalic roof to produce the elongated stalk and photoreceptive endvesicle, the parapineal remains a rudimentary structure, persisting near the base of the pineal stalk closely apposed to the left habenula [62]. Size differences between the habenulae are less prominent in zebrafish than in primitive fish or Rana, with the left habenula of 4-day-old zebrafish larvae being , 15 – 20% larger than that on the right [62]. More impressive are the dense neuropil of the left habenula and expanded expression of the recently identified leftover (lov) gene [60,62]. Zebrafish lov encodes an unknown protein that contains an oligomerization domain similar to Shaker-related Kþ channel subunits [63]. lov is transcribed throughout the left habenula at high levels, but at lower levels and in a limited region on the right. Expression is asymmetric from its onset and persists in this pattern throughout life [62]. Left-sided expression of the Nodal pathway in the embryonic pineal, the left-positioned parapineal, and the enlarged neuropil and lov expression in the left habenula are found in the vast majority of zebrafish, indicating a strong population-related bias to maintain directional asymmetry of the epithalamus. Global regulation of left– right asymmetry Lessons learned from studies of visceral asymmetry are useful for understanding how left –right differences might be set up in the brain. Many mutations in mouse and zebrafish are known to cause left–right randomization (antisymmetry) in which, for example, the normally rightward
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Fig. 2. Sequential appearance of epithalamic asymmetry. (a) In the segmenting zebrafish embryo (20 hours post-fertilization), cyclops/nodal-related 2 (cyc/ndr2, blue), antivin/lefty (lft1) and pitx2 (not shown) are briefly transcribed on the left side of the presumptive pineal, which is defined by its bilateral expression of the floating head gene (red) [79] (i). By two days post-fertilization, the parapineal (arrowhead) has formed to the left of the pineal, as revealed by otx5 expression (ii). In four-day-old larvae, the dorsal habenular nuclei express the leftover gene (iii) and exhibit differences in neuropil density (iv) in a characteristic left –right pattern. (b,c) Schematic of left– right differences in the epithalamic development of wild-type (b) and mutant (c) zebrafish. Mutations that perturb midline formation or Nodal signaling result in bilateral or absent pineal gene expression, respectively. In both classes of mutants, parapineal sidedness and habenular laterality are left –right randomized, but always in a concordant manner. All panels show dorsal views.
looping of the heart tube is reversed in half of affected individuals [24,25]. In zebrafish, defects in visceral asymmetry result from a variety of perturbations, including defective tissue patterning at gastrulation, abnormal midline formation and loss of Nodal pathway components [45–51]. The same early-acting mechanisms also regulate epithalamic asymmetry. However, some zebrafish mutants show discordance in the direction of visceral and brain laterality [45,49–51], demonstrating that there are both global and tissue-specific aspects to left–right determination. With respect to global left– right specification, it is still unresolved what the first symmetry-breaking event is and whether it occurs and is propagated similarly in all vertebrates. Recent work in Xenopus implicates asymmetric Hþ and Kþ fluxes and directional gap-junction communication as very early components of left– right patterning that lead to asymmetric nodal expression in the lateral-plate mesoderm [64]. Genetic and cellular studies in mice support a monocilia-mediated mechanism that initiates molecular left– right asymmetry through the directional movement or perception of signals. The socalled nodal flow generated by monociliated cells in the embryonic node is disrupted in mouse mutants showing left –right defects [24]. Although epithelial cells lining Kupffer’s vesicle in the zebrafish also possess motile cilia [65], this structure probably develops too late to qualify as the initiator of left– right axis formation. Sequential development of epithalamic laterality Epithalamic asymmetry arises sequentially in zebrafish and appears to be intimately connected with morphogenesis of the pineal complex. Some insight into this process came from mutants that have disrupted midline formation and http://tins.trends.com
that later express cyc, lft1 and pitx2 on both sides of the pineal anlage [45 –51]. Their parapineal develops either to the left or right of the pineal and is only rarely (, 1%) duplicated bilaterally. In half of such mutant larvae, the neuropil and lov habenular asymmetries are left –right reversed in parallel [60,62]. By contrast, mutations that inactivate Nodal signaling or reception inhibit left-sided gene expression in the pineal anlage [60,62]. This finding allows the function of the Nodal diencephalic asymmetry to be assessed. For example, mutants lacking the cofactor One-eyed pinhead can be rescued past the requirement for Nodal signaling at gastrulation by injection of oep RNA at the one-cell stage [56]. Injected embryos later lack left-sided gene expression in the lateral-plate mesoderm and pineal anlage, yet they develop into adult fish [60,62,66]. In the brains of rescued oep adults, the pineal stalk frequently emerges from the diencephalic roof at an inappropriate position relative to the usual location just left of the midline (Fig. 3). Asymmetric Nodal signaling is not required for morphogenesis of the pineal organ but, rather, it positions outgrowth of the anlage along the left– right axis of the diencephalon [49]. In addition, Nodal signaling influences parapineal position and habenular laterality [60 – 62]. As in midline mutants, half of rescued oep larvae display a right-sided parapineal and left – right reversal of habenular asymmetry (Fig. 2). Therefore, bilateral activation and absence of Nodal activity in the pineal anlage have the same consequence, randomizing the directionality of epithalamic asymmetry across the population. Regardless of sidedness, laterality of the parapineal and habenulae always correspond, suggesting that they are
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Fig. 3. Left –right displacement of the pineal stalk. (a) In adult zebrafish, the pineal stalk usually emanates from the dorsal diencephalic roof with a slight left bias. (b) The left– right position is frequently shifted in mutants that lacked Nodal signaling in their embryonic pineal anlage. (c) Summary diagram showing the midpoint of the pineal stalk relative to the midline of the adult brain. Rescued one-eyed pinhead (oep) mutants (pink circles) show a greater variation in pineal organ placement along the left– right axis of the diencephalon than wild-type zebrafish (blue circles).
coordinately regulated [60,62]. Alternatively, the parapineal itself could modulate left– right identity of the adjacent habenula. In fact, axonal tracing studies demonstrate that in some species the parapineal preferentially innervates the left habenula [28,67]. Strong support for a direct role for the parapineal in habenular laterality comes from laser ablation experiments (Fig. 4). After selective destruction of the parapineal, both habenulae develop but the left habenula is indistinguishable from the right. Conversely, in rare cases where parapineal organs are duplicated to the left and right of the pineal, both habenulae adopt the denser neuropil and extensive lov expression characteristic of the left habenula (i.e. there is left isomerization) [62]. The zebrafish studies promote a model in which global signals set up a left –right bias in cyc, lft1 and pitx2 transcription in the embryonic diencephalon, as they do in the left lateral-plate mesoderm. Asymmetric activation of flh:GFP
the Nodal signaling pathway regulates laterality of the pineal complex, normally directing the parapineal to form to the left of the pineal anlage. The left-sided parapineal promotes the differentiation of left-specific properties in the adjacent brain nucleus, an interaction that potentially could persist in the adult brain. An alternative interpretation, which has not been ruled out, is that the parapineal serves to reinforce or maintain a predetermined laterality in the habenular region. In either case, the nature of the interaction between the parapineal and left habenula is unknown, although close proximity between the two structures appears to be necessary. The strength of the zebrafish system is its amenability to mutagenesis and to genetic dissection of developmental processes [68]. As first demonstrated for hindbrain segmentation [69], mutational screening of the basis of gene expression patterns is readily performed, yielding specific mutations that cause subtle alterations that are not detected in morphology screens. Such an approach using lov should identify early-acting genes that regulate laterality of the brain and viscera as well as those controlling brain-specific events. Significance of epithalamic asymmetry Countless questions remain concerning the importance and universality of diencephalic asymmetry. Laterality of the zebrafish epithalamus is robustly preserved in individuals and at the population level (99% of larvae develop epithalamic asymmetry and 95% have the leftbiased pattern), implying that directional asymmetry confers an advantage for the species. Left– right preferences in eye usage [70– 73] and directional biases in swimming in some teleost fish [74] are thought to mediate social behaviors such as schooling, feeding and the escape response. Whether diencephalic asymmetry is required for expression of lateralized larval and adult behaviors is presently unknown; however, mutant strains should permit rigorous testing of the correlation between anatomy and function. Left– right differences in the habenulae are likely to have an impact on other brain regions. As reported for some fish and frogs, left –right habenular efferents of the fasciculus retroflexus could vary [35,67],
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Fig. 4. The parapineal mediates habenular asymmetry. The pineal and parapineal can be visualized in vivo in floating head: green fluorescent protein ( flh:GFP) transgenic embryos [62], allowing selective laser-mediated cell ablation (red cross). Ablation of cells to the right of the pineal complex (control ablated) does not affect the later expression of symmetric (cpd2, gray) or asymmetric (lov, red) habenular markers, or expression of otx5 in the pineal complex (blue). By contrast, the expanded lov expression pattern and denser neuropil (not shown) characteristic of the left habenula do not appear following destruction of the parapineal (confirmed by loss of the otx5 parapineal domain). In its size and molecular properties, the left habenula resembles the right in parapineal-ablated larvae. http://tins.trends.com
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leading to differential synaptic activity at the target interpeduncular nucleus. Telencephalic inputs might differ if left– right habenulae express different molecular cues when connections are established. Zebrafish transgenic lines should allow direct visualization of the habenular circuitry through spatially regulated expression of green fluorescent protein (GFP) variants. A pressing issue is whether the zebrafish studies will reveal general strategies for constructing brain asymmetry and, thus, provide clues about the evolutionary events that set the framework for the sophisticated hemispheric specialization of the human cortex. Anatomical asymmetry of the diencephalon is an ancient property of the vertebrate lineage that seems more prominent in evolutionarily lower taxa [28], perhaps reflecting a diminished need for its preservation as laterality became fixed and complexity increased in other forebrain regions. Neuroanatomical asymmetry is also found outside the cortex, in thalamic nuclei, the basal ganglia and aspects of the neuroendocrine system [75– 77]. An added complication is that asymmetric morphologies are not exclusively under genetic control but are also the product of hormonal regulation, environmental conditions and experience [23]. Despite intensive searching, there is no evidence yet that the Nodal pathway functions unilaterally in the embryonic brain of mouse, chick or Xenopus, even though Nodal activation in the left lateral-plate mesoderm is conserved. In mammals, molecular left –right differences might be transient or confined to small groups of neurons and, thus, challenging to uncover. The identification of genes such as lov, which is expressed in the zebrafish habenulae throughout life and belongs to a vertebrate multi-gene family [62], could be an asset for comparative studies. Whole genome approaches, such as microarray technology, that utilize probes derived from left-sided versus right-sided brain tissue might prove sensitive enough to detect other differentially expressed genes [78]. From studies of the photoreceptive pineal complex and habenulae, Kemali proposed that left– right asymmetry was ‘imposed onto the brain from the periphery’ and contemplated whether lateralized brain functions had their evolutionary origin in the anatomical asymmetry of the epithalamus [27]. Genetic screens in model systems and the application of molecular methods to diverse species provide a unique opportunity to explore the development, conservation and function of diencephalic asymmetry. Acknowledgements We are grateful to Christine Thisse and Bernard Thisse for generously providing zebrafish brain markers and to Chris Wright and Luis Puelles for valuable discussions. Described research was supported in part by the NSF (M.E.H.) and by post-doctoral fellowships from NRSA (J.O.L.) and the American Cancer Society (J.T.G.).
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