Research article 4869 Directional asymmetry of the zebrafish epithalamus guides dorsoventral innervation of the midbrain target Joshua T. Gamse1,*,†, Yung-Shu Kuan1,†, Michelle Macurak1, Christian Brösamle1, Bernard Thisse2, Christine Thisse2 and Marnie E. Halpern1,‡ 1Carnegie Institution of Washington, Department of Embryology, 3520 San Martin Drive, Baltimore, MD, 21218, USA 2IGBMC, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP10142, CU de Strasbourg, 67404 Illkirch cedex, France *Present address: Vanderbilt University, Department of Biological Sciences, VU Station B, Box 35-1634, Nashville TN 37235-1634, USA †These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 9 August 2005

Development 132, 4869-4880 Published by The Company of Biologists 2005 doi:10.1242/dev.02046

Summary The zebrafish epithalamus, consisting of the pineal complex channel tetramerization domain containing) gene family are and flanking dorsal habenular nuclei, provides a valuable expressed differently by the left and right habenula, in model for exploring how left-right differences could arise patterns that define asymmetric subnuclei. Molecular in the vertebrate brain. The parapineal lies to the left of the asymmetry extends to protein levels in habenular efferents, pineal and the left habenula is larger, has expanded dense providing additional evidence that left and right axons neuropil, and distinct patterns of gene expression from the terminate within different dorsoventral regions of the right habenula. Under the influence of Nodal signaling, midbrain target. Laser-mediated ablation of the parapineal positioning of the parapineal sets the direction of habenular disrupts habenular asymmetry and consequently alters the asymmetry and thereby determines the left-right origin of dorsoventral distribution of innervating axons. The results habenular projections onto the midbrain target, the demonstrate that laterality of the dorsal forebrain interpeduncular nucleus (IPN). In zebrafish with influences the formation of midbrain connections and their parapineal reversal, neurons from the left habenula project molecular properties. to a more limited ventral IPN region where right habenular Development axons would normally project. Conversely, efferents from the right habenula adopt a more extensive dorsoventral Key words: KCTD gene family, leftover gene, Left-right asymmetry, IPN projection pattern typical of left habenular neurons. Habenular nuclei, Fasciculus retroflexus, Interpeduncular nucleus, Three members of the leftover-related KCTD (potassium Diencephalon

Introduction parapineal transcribe genes encoding melatonin biosynthetic A growing number of studies has dispelled the notion that enzymes in a circadian-regulated manner (Gamse et al., 2003; lateralization of the brain is unique to the human cortex (see Gothilf et al., 1999), but the specific role of the parapineal is Rogers and Andrew, 2002). Even in the Drosophila brain, unknown. The bilateral habenular nuclei, which flank the structural asymmetry has been linked to long-term memory pineal complex, can differ in size and structure (Harris et al., (Pascual et al., 2004), and laterality has been well described in 1996; Concha and Wilson, 2001). In some amphibians, the left the nematode primitive sensory nervous system (Chang et al., habenula contains two morphologically distinct subdomains 2004; Chuang and Bargmann, 2005). From fish to primates, whereas there is only a single nucleus on the right (Wehrmaker, evidence has accumulated for left-right (L-R) differences in 1969; Braitenberg and Kemali, 1970; Morgan et al., 1973). The brain anatomy, cellular organization, ultrastructure and degree of habenular asymmetry can vary seasonally in frogs, neurotransmitter distribution and, in a few cases, asymmetry presumably correlated with the mating period (Kemali et al., has been linked to behavior (Rogers and Krebs, 1996; Skiba et al., 2002; Toga and Thompson, 2003; Vallortigara et al., 1999). 1990). In chickens, L-R habenular differences are sex- Despite significant progress, our understanding of the dependent and influenced by hormonal levels (Gurusinghe and development and evolution of lateralized features of the Ehrlich, 1985; Gurusinghe et al., 1986). vertebrate nervous system remains limited. The habenulae relay impulses from limbic areas of the In lower vertebrates, the epithalamus of the dorsal telencephalon to an unpaired midbrain nucleus, the diencephalon displays notable asymmetries (Concha and interpeduncular nucleus (IPN) via the fasciculus retroflexus Wilson, 2001; Halpern et al., 2003). The epithalamus includes (FR) nerve bundle (of Meynert), in a conduction system that is the pineal complex, which in many fish consists of the pineal conserved evolutionarily (Sutherland, 1982). The function of organ and an asymmetrically positioned accessory organ the habenulointerpeduncular connection is poorly understood, termed the parapineal (Borg et al., 1983). Both the pineal and although there is evidence from rodents that it modulates 4870 Development 132 (21) Research article

complex behaviors such as avoidance, reward and feeding Lov+ and Ron+ immunoreactive axons, demonstrates that L-R (Sutherland, 1982). habenular efferents project to different regions along the Progress in determining how epithalamic asymmetry arises dorsoventral (DV) axis of the IPN. Reversal of habenular and the molecular pathways involved has come from studies in laterality by perturbation of Nodal signaling also reverses the zebrafish. cyclops (cyc), which encodes a zebrafish Nodal- L-R origin of projections onto the IPN. Ablation of the related Tgf␤ signal (Rebagliati et al., 1998a; Rebagliati et al., parapineal disrupts laterality and the stereotypic DV pattern of 1988b; Sampath et al., 1998), and other genes functioning in IPN connectivity. The results demonstrate that L-R differences this signaling pathway, are expressed transiently on the left side of the dorsal forebrain influence the connections and molecular of the embryonic pineal anlage (Bisgrove et al., 2000; Concha properties of innervating axons at the midbrain target. et al., 2000; Liang et al., 2000). Nodal signals are known to mediate laterality of the heart and visceral organs in vertebrate Materials and methods embryos and play an important role in gastrulation (Schier, Zebrafish 2003). Zebrafish mutants for one-eyed pinhead (oep), which Zebrafish were raised at 28.5°C and staged according to hours or days encodes an obligatory component of the Nodal receptor post-fertilization. AB wild-types (Walker, 1999), transgenic lines complex, can be rescued past the early requirement for Nodal Tg(flh:GFP)c161, Tg(flh:GFP)c162 (Gamse et al., 2003) and signaling by injection of oep RNA (Yan et al., 1999; Zhang et Tg(foxd3:GFP) (Gilmour et al., 2002) and mutants carrying the null al., 1998). Rescued embryos (Roep) lack asymmetric gene allele oeptz257 (Brand et al., 1996) were used. Embryos completely expression in the embryonic pineal but develop to adulthood, lacking maternal and zygotic Oep function (MZoep) (Yan et al., 1999) thus allowing Nodal pathway function in the brain to be were generated from homozygous mutant adults produced by rescuing tz257 assessed (Concha et al., 2000; Liang et al., 2000). In Roep fish oep homozygous 1- to 2-cell stage embryos with injection of sense the directionality of L-R differences in diencephalic anatomy oep RNA (80 pg). RNA was synthesized from pCS2-oep (Liang et al., is randomized (Concha et al., 2000; Liang et al., 2000; Gamse 2000) using the SP6 mMessage mMachine Kit (Ambion). et al., 2002). The parapineal, located to the left of the pineal Morpholino injections anlage in >95% of wild-type (WT) larvae, develops to the right Antisense spaw morpholino oligonucleotides (MO) were derived from in approximately half of Roep larvae and the asymmetric the splice acceptor site of the last intron (Spaw-MO2) (Long et al., properties of the habenular nuclei, which include differences 2003). MO stock solution (10 mg/ml) was diluted (6 ng/nl in dH2O) in size, neuropil density and gene expression, are L-R reversed. and Tg(foxd3:GFP) embryos (1- to 2-cell stage) were pressure injected with 1 nl to deliver 6 ng of MO. Larvae were presorted For example, the leftover (lov) gene is typically transcribed by + more cells of the left habenula than the right; however, half of according to whether the GFP parapineal was situated to the left or Roep larvae show the opposite pattern (Gamse et al., 2003). right of the pineal anlage. Over 90% of MO-injected embryos hatched and, at 4 days, were used for whole-mount in situ hybridization or Parapineal L-R position always corresponds with the direction antibody labeling with anti-acetylated tubulin antibody (Concha et al., of habenular laterality. Moreover, following parapineal 2000; Gamse et al., 2003). Others were raised to adulthood for brain Development ablation, the habenulae fail to develop asymmetrically (Concha dissection and hodological analysis. et al., 2003; Gamse et al., 2003). Even earlier in development, signaling by another Nodal- Dye labeling of the habenulae related factor, Southpaw (Spaw), influences asymmetric gene After anesthetization with tricaine (170 ␮g/ml), 2- to 3-month-old expression in the zebrafish diencephalon. spaw is the earliest adults (AB) were decapitated with a razor. Brains were dissected out known gene to be expressed unilaterally, with transcripts in cold phosphate-buffered saline (PBS), pinned dorsal side upwards on Sylgard (Molecular Probes)-coated dishes, and fixed in 4% appearing in the left lateral plate mesoderm (LPM) by the 10- paraformaldehyde for at least 24 hours at 4°C. Lipophilic dyes FAST 12 somite stage (Long et al., 2003). However, expression is not DiI and FAST DiO (Molecular Probes) were dissolved in detected in the embryonic brain. Unlike the zebrafish Nodal- dimethylformamide at 50 mg/ml by heating at 50°C for 5 to 10 related signals, Cyc and Squint (Sqt), that mediate tissue minutes. Aliquots of the dye solutions were stored at –80°C, thawed specification in the early embryo (Hatta et al., 1991; Feldman and briefly heated at 42°C before backloading into glass needles. Prior et al., 1998), Spaw appears to regulate the L-R axis specifically. to dye application, the prominent commissure that extends between Embryos deficient for Spaw lack cyc, pitx2 and lft1 expression the left and right habenulae was severed with a tungsten needle to in the left diencephalon (Long et al., 2003), and thereby affect prevent dye passage. Each habenula was impaled and dye applied morphological asymmetry of the epithalamus. The zebrafish using a pressure injector (Aizawa et al., 2005). Labeled brains were studies suggest that brain laterality results from a cascade of stored in 4% paraformaldehyde at 28°C for 18-21 days because of the distance the lipophilic dyes must travel along the habenula FR developmental events that leads from left-sided Nodal projections to reach the IPN. Dye passage was monitored under a signaling in the pineal anlage to L-R assignment of parapineal Leica MZFLIII stereomicroscope. Labeled brains were embedded in position, which, in turn, directs habenular asymmetry. low-melting temperature agarose (4%) in plastic molds for transverse Here, we provide additional evidence for molecular L-R vibratome (Leica VT1000S) sectioning. Sections (100-150 ␮m) were differences in the zebrafish habenular nuclei, which extends to mounted on glass slides in cold Mowiol (Calbiochem) medium (480 their efferent projections and innervation of the midbrain mg/ml Mowiol in 25% glycerol with 0.1 M Tris, pH 8.5) and imaged target, the IPN. Two genes related to lov, right on (ron) and with a Leica MZFLIII or SP2 confocal microscope. dexter (dex), are also expressed asymmetrically in the Identification of leftover-related genes habenular nuclei but to a greater extent on the right than the Individual clones from an adult zebrafish kidney cDNA library left. Consequently, Lov and Ron proteins are distributed containing inserts between EcoRI and XhoI sites of pBK-CMV differently in habenular subdomains and in efferent axons (Stratagene) were assayed for expression during embryonic stages within the FR on the left and right sides of the larval brain. by whole-mount in situ hybridization. A partial ron cDNA was Differential dye-labeling of adult habenulae, or visualization of isolated and the transcript 5Ј end identified by RNA-ligase RT-PCR Asymmetry of habenular projections 4871

(RLM-RACE Kit, Ambion) using total RNA from 4-day larvae. approximately 15-20 cells ablated by 5-10 pulses/cell from a 440 nm Scanning of the zebrafish genome database laser beam (Photonic Instruments) focused through a 40ϫ water (Zv3;http://www.sanger.ac.uk/Projects/D_rerio/) for sequences immersion objective mounted on a Zeiss Axiophot microscope. An similar to lov and ron, yielded dexter (dex) and homologues of equivalent number of cells contralateral to the parapineal were ablated KCTD12b, 16a and 16b. Primers specific for dex, kctd12b, 16a and in controls. 16b sequences were used to amplify the open reading frame by RT- PCR (Retroscript Kit, Ambion), products were subcloned into pCRII Accession numbers (Invitrogen), and confirmed by sequencing. kctd16a, kctd12b and GenBank accession numbers: KCTD16a, AY763407; KCTD12b, dex map to the same arm of linkage group 14. AY763408; KCTD16b, AY763409; dex, AY763410; ron, AY763411. RNA in situ hybridization Protocols were followed as in Gamse et al. (Gamse et al., 2002), using Results reagents from Roche Molecular Biochemicals. To synthesize A family of lov-related genes antisense digoxigenin or fluorescein RNA probes, insulin (Stafford and Prince, 2002), pBS-otx5 and pBK-CMV-lov were linearized with The leftover gene was previously identified in a screen of random EcoRI and transcribed with T7 RNA polymerase, pBK-CMV-ron with cDNA clones for tissue-specific expression in zebrafish embryos BamHI and T7 RNA polymerase, pCRII-KCTD16a and pCRII- and larvae. lov expression was found to differ on the left and right KCTD12b with SpeI and T7 RNA polymerase, and pCRII-KCTD16b sides of the diencephalon, in a region corresponding to the paired and pCRII-dex with XhoI and SP6 RNA polymerase. Hybridized habenular nuclei (Gamse et al., 2003). probes were detected using alkaline phosphatase-conjugated Upon subsequent screening, another gene, right on (ron), antibodies and visualized by 4-nitro blue tetrazolium (NBT) and 5- was found to show asymmetric expression in the habenulae. bromo-4-chloro-3-indolyl-phosphate (BCIP) staining for single Remarkably, ron shares a high degree of sequence homology labeling, or NBT/BCIP followed by iodonitrotetrazolium (INT) and with lov, particularly in two discrete domains of the predicted BCIP staining for double labeling. Larvae were embedded in 4% low- protein coding sequence (Fig. 1A). Scanning of available melt agarose (Cambrex Rockland, ME) for vibratome sectioning (100-150 ␮m), or in London Resin Gold (Ted Pella, Redding, CA) zebrafish genomic sequence and degenerate PCR yielded four and sectioned using a Reichert ultramicrotome (8-10 ␮m) followed additional homologous genes (Fig. 1A). Only one, dexter (dex), by Basic Fuchsin (Sigma) counterstaining. is expressed in the habenulae (see below). In 4-day larvae, ron is also expressed in the gall bladder and posterior border of the Lov and Ron antibodies optic tectum, and dex in paired groups of cells in the ventral Partial lov and ron open reading frames were subcloned into diencephalon (data not shown). pDEST17 (Invitrogen), which adds a 6-histidine tag to the amino (N) Related genes from mammals were grouped as the terminus. Tagged protein was not recovered at high levels from potassium channel tetramerization domain containing (KCTD) bacterial cells unless the T1 domain was also deleted. T1-deleted constructs were expressed in BL21(DE3)pLysS bacteria (Stratagene), gene family (Marchler-Bauer et al., 2003). Structurally, KCTD proteins contain a N-terminal sequence homologous to the T1

Development the protein purified on nickel-NTA columns (Qiagen) and injected into rabbits (Spring Valley Laboratories) to produce polyclonal tetramerization domain of the Shaker class of voltage-gated antibodies against Lov or Ron. Resultant sera were purified using potassium channels (Papazian, 1999). However, they have no HiTrap affinity columns (Amersham). Specificity was confirmed by other features of channel proteins, such as transmembrane western blotting using standard methods (Gallagher et al., 1989) and domains. Phylogenetic comparison with human and mouse by immunofluorescence in embryos injected with lov or ron genes reveals that the zebrafish proteins (Fig. 1B, boxed) fall morpholinos. into four subclasses of the larger KCTD family: Kctd16a/b; Immunofluorescence Kctd12b; Lov/Ron/Kctd12/Pfetin and Dex/Kctd8. The Four-day larvae fixed overnight in 4% paraformaldehyde were stored zebrafish kctd12b, 16a and 16b genes are strongly expressed in methanol at –20°C for up to 2 weeks. Following rehydration in the retina. Additionally, kctd16a is expressed in the dorsal through a methanol/PBS series, samples were permeabilized with forebrain and optic tectum and kctd16b in olfactory epithelium proteinase K (10 ␮g/ml; Roche) for 30 minutes, refixed, blocked in (data not shown). PBS/0.1%TritonX-100/10% sheep serum (PBSTS), and incubated In humans, KCTD12/PFET expression was detected in the overnight in primary antibody diluted 1:500 in PBSTS. Primary fetal cochlea (Resendes et al., 2004), a structure absent in the antibody was detected using goat-anti-rabbit:Cy3 (Jackson zebrafish (Whitfield et al., 2002). We found that Kcdt12/Pfet is Immunoresearch) or goat-anti-rabbit:Alexa Fluor 488 (Molecular also expressed in the medial habenulae of the fetal mouse brain Probes). For double labeling with Lov and Ron antibodies, the first and Kcdt8 transcripts specifically localize to the medial primary was detected with goat-anti-rabbit Fab fragment:Rhodamine (Jackson Immunoresearch), then blocked with unlabeled goat-anti- habenulae of the adult rat brain (Fig. S1 in supplementary rabbit antibody. The protocol for the second primary was the same as material). single labeling. Larvae were mounted in glycerol and images collected on a Leica SP2 confocal microscope. For vibratome sectioning, brains Left-right differences in habenular gene expression of adult fish derived from spaw MO-injected embryos or WT controls patterns were dissected, fixed overnight at 4°C and embedded as above. Zebrafish ron and dex are expressed asymmetrically in the Sections were arranged on precleaned Superfrost slides (VWR), dried habenular nuclei; however, in contrast to lov, they are briefly and double labeled with Lov and Ron antibody. transcribed in more cells of the right habenula than the left. Laser-mediated cell ablation Expression, which is asymmetric from the outset, is first Parapineal ablation was performed using Tg(flh:GFP)c161, detected at 2 days (Fig. 1C,G), and increases significantly by Tg(flh:GFP)c162 or Tg(flh:GFP)c161/+;Tg(foxd3:GFP)/+ 28- to 32- 4 days (Fig. 1D,H). Parasagittal sections reveal that ron and hour-old embryos, mounted dorsal side up in 1.2% agar on glass dex are expressed in the dorsal region of the right but not the slides. The parapineal was visualized by GFP fluorescence and left habenula (compare Fig. 1E and I with F and J). Expression 4872 Development 132 (21) Research article

of dex extends more laterally in both habenulae than expression demonstrated that protein levels differ between the left and right of ron (Fig. 1D,H). habenulae in a pattern closely reflecting mRNA distribution Double label in situ hybridization allows further refinement (Fig. 2A-C). Although transcripts were restricted to the cell of habenular gene expression subdomains (Fig. 1K-N, and not bodies of habenular neurons, protein was also detected within shown). Only cells in the dorsal region of the left habenula ventrocaudally projecting axons that course through the FR, and express lov, while those in the ventral region express ron and in synaptic terminals at the midbrain target, the interpeduncular dex. In contrast, cells in both dorsal and ventral regions of the nucleus. Lov+ growth cones reach the IPN by 2 days (Fig. 2D). right habenula express lov, ron and dex. All three genes are By 4 days, habenulointerpeduncular connections are well transcribed in medial and posterior regions of the right formed: the Lov+ axonal bundle is larger within the left FR than habenula, but only ron and dex expression is detected in the right, and Lov+ (Fig. 2E,F) and Ron+ (Fig. 2G) habenular anterior and lateral regions. efferents project extensively along the IPN. Innervation of the target was distinguished more clearly in lateral views of the Molecular L-R specialization of larval brain. Lov+ axons project to the dorsal and ventral IPN habenulointerpeduncular connections (Fig. 2I), while most, if not all, Ron+ axons project to the ventral To determine whether asymmetric gene expression leads to IPN (Fig. 2J). Double labeling confirmed that Lov+ axons L-R differences in protein distribution, and to evaluate traverse the dorsal and ventral regions of the midbrain target subcellular localization, we generated polyclonal antibodies and Ron+ axons are confined ventrally (Fig. 2K). against Lov and Ron. Rabbit sera reacted specifically against each protein on western blots and in Lov or Ron MO-depleted Epithalamic L-R asymmetry guides DV innervation embryos (Fig. S2 in supplementary material). of midbrain target Confocal microscopy of Lov and Ron immunolabeled larvae Parapineal position was shown to correlate with higher levels

Fig. 1. leftover (lov)-related genes are asymmetrically expressed in zebrafish diencephalon. (A) Five zebrafish proteins, Right on (Ron), Dexter (Dex), and Potassium channel tetramerization domain- containing proteins 12b (Kctd12b), 16a and 16b, are structurally similar to Lov (% amino acid identity indicated), contain an N-terminal T1

Development domain related to the tetramerization domain of the Shaker potassium channel (blue box) and a conserved C-terminal domain (red box) (see Gamse et al., 2003). (B) Zebrafish Lov-related family members (boxed) and homologous mammalian proteins fall into four major groups (designated by colors). The unrooted phylogenetic tree was calculated using the CLUSTAL_X algorithm (Thompson et al., 1997). Scale bar indicates a 10% difference in amino acid identity, measured along the shortest path between two proteins. Dr, Danio rerio; Mm, Mus musculus; Hs, Homo sapiens. (C,D,G,H) Asymmetric expression of (C) ron and (G) dex in habenular nuclei is detected at 2 days, and increases significantly by 4 days (D,H). Parasagittal sections through (E,I) left (Lh) and (F,J) right (Rh) habenulae reveal ron and dex transcripts throughout Rh, but confined to more ventral regions of Lh (dorsal regions outlined in E and I). (K-M) Double in situ hybridization shows lov and ron DV expression is largely non-overlapping in Lh (L) but coextensive in Rh (M). (N) dex habenular expression partially overlaps with lov, although most dex-expressing cells in Lh are more ventral than lov-expressing cells. C,D,G,H,K and N are dorsal views with anterior to the top; scale bar: 50 ␮m. Left (L) and right (R) sides of the brain and midline, indicated in C, apply to all dorsal views. Arrows in D,H and K indicate approximate position of sections shown in subsequent panels. E,F,I and J are parasagittal plastic sections (8-10 ␮m) with dorsal to the top and anterior to the left; scale bar: 20 ␮m. L and M are vibratome sections (100 ␮m) with dorsal to the top and anterior to left; scale bar: 30 ␮m. Asymmetry of habenular projections 4873

of lov RNA in the adjacent habenula of normal or L-R reversed Roep brains (Gamse et al., 2003) (Fig. 3A,B). Conversely, ron and dex expression is more extensive in the habenula opposite the parapineal (Fig. 3C-F), irrespective of directionality. To determine whether projections from the habenula to the IPN are also influenced by the L-R position of the parapineal and direction of habenular asymmetry, we examined Lov and Ron immunoreactivity in Roep larvae. In agreement with transcriptional patterns, the asymmetric distribution of Lov and Ron proteins in the habenulae and FR was similar to WT in half of Roep larvae (n=39, Fig. 3G,I). The other half (n=31) showed a L-R reversal in protein levels (Fig. 3H,J) that extended to habenular efferents within the FR. We examined whether altered habenular laterality affected innervation along the DV axis of the IPN. Lov+ efferents project to the dorsal and ventral IPN, while Ron+ fibers innervate the ventral IPN in Roep larvae that have a left-positioned parapineal (Fig. 3K) and those that are L-R reversed, with the parapineal on the right (Fig. 3L). In such larvae, however, input to the IPN shows a mirror image reversal (Fig. 3H). Thus, when the L-R origin of habenular efferents is opposite that of WT, the DV pattern of immunoreactive connections to the IPN is still preserved. Development Southpaw regulates directional asymmetry of habenulo- interpeduncular projections Targeted depletion of Spaw in zebrafish Fig. 2. Asymmetric distribution of Lov and Ron in habenular neurons and their embryos was previously shown to alter projections. (A-C) Immunofluorescent detection of (A) Lov and (B) Ron proteins and laterality of the heart and pancreas and (C) colocalization in the habenular nuclei correlates well with the pattern of prevent expression of Nodal pathway genes in transcriptional domains (see Fig. 1). (D) Growth cones of Lov+ habenular efferents in the left LPM and diencephalon (Long et al., FR reach the ventral midbrain by 2 days. (E) By 4 days, asymmetric Lov+ habenular projections traverse the target IPN. (F,I) Lov+ and (G,J) Ron+ axons project to different 2003). Because disruption of diencephalic + gene expression causes L-R randomization of domains along dorsoventral axis of IPN. (I) Lov neurons terminate in anterior epithalamic asymmetry (Liang et al., 2000; (asterisk) and posterior (closed arrowhead) regions of dorsal IPN and in ventral IPN (open arrowheads); (J) most Ron+ neurons project to ventral IPN. (Ron+ Gamse et al., 2003; Concha et al., 2000; immunofluorescence at dorsal IPN was barely distinguishable from background levels, Concha et al., 2003), lack of Spaw should also which were always higher than observed with the Lov antiserum.) All images are affect directional asymmetry of the pineal confocal Z-stacks from (A-H) the dorsal aspect, anterior to top, or (I-K) the lateral complex and habenular nuclei. aspect, anterior to left. Scale bars: 30 ␮m (A-C,F-K); 50 ␮m (D,E). spaw MO was injected into embryos bearing the foxD3:GFP transgene (Gilmour et al., 2002), which is highly expressed in the pineal complex at embryos had L-R reversals in parapineal position and 2-3 days (Concha et al., 2003) and allows unambiguous habenular asymmetry (45%, n=418). A smaller number (3%) assignment of the L-R position of the parapineal relative to the exhibited bilaterally symmetric lov expression in the midline pineal, throughout larval stages (Fig. S3A,B in habenulae. These findings confirm that signaling by Spaw supplementary material). The direction of habenular regulates directional asymmetry of the developing asymmetry was determined at 4 days by assessing L-R epithalamus. differences in neuropil density (Fig. S3C,D in supplementary The endocrine pancreas normally originates to the right of material) or lov expression (Fig. S3E,F in supplementary the midline in WT embryos (see Stafford and Prince, 2002). material). As summarized in Table 1, Tg(foxD3:GFP) controls About twice as many spaw MO-injected larvae developed did not show reversals in habenular laterality (n=300). the pancreas on the right side than on the left (Fig. S3GI and However, almost half of larvae derived from spaw MO-injected H,J in supplementary material), irrespective of the 4874 Development 132 (21) Research article

directionality of brain asymmetry. The data provide further heterotaxia in a significant fraction of the population, the support to the proposal that Spaw signaling coordinates majority of Spaw-depleted embryos hatched, developed swim directional asymmetry of the visceral organs and the brain bladders (>90%) and survived to adulthood (>60%). Injection and, that in the absence of this coordination, L-R reversal of of spaw MO into foxD3:GFP embryos was therefore a useful brain and pancreas laterality occurs in a largely independent method for generating larvae with L-R randomized fashion. diencephalic asymmetry that could be screened on the basis of Despite alterations in the L-R axis and presumptive a left-sided or right-sided parapineal, separated, and raised to adulthood. This allowed habenular projections to be traced in the brains of adult fish that had a pre-determined parapineal position. Habenular efferents and their connections were traced (as in Fig. 4A,B) by uniquely labeling each habenula with the lipophilic dyes DiO (left side) or DiI (right side). As was previously described for adult zebrafish (Tomizawa et al., 2001), anterograde DiI labeling of both habenulae reveals efferents that course posteriorly through the fasciculi retroflexus to the midline IPN in the ventral midbrain. Although differentially labeled efferents from the right and left habenulae of adult fish follow a similar trajectory (Fig. 4C-I), they project to different regions along the DV axis of the IPN (Fig. 4J-L). The left habenula innervates the dorsal and ventral IPN (Fig. 5A,C) and the right habenula innervates only the ventral region (Fig. 5B,C). In adult fish derived from spaw MO-injected embryos, the brains from those that had formed the parapineal on the left side (n=4) (Fig. 5D-F) showed an IPN innervation pattern similar to WT. Brains with the parapineal on the right (n=4) exhibited a reversed pattern, in which efferents from the left habenula (green) projected solely to the ventral region of the IPN and those from the right habenula projected along the entire DV axis (Fig. 5G-I). Similar to Roep larvae, the overall DV pattern of efferents was preserved in adult brains even Development when the habenula of origin was L-R reversed. The adult projection pattern onto the IPN was invariant in WT fish and closely paralleled by the distribution of Lov and Ron protein in habenular axons. Lov+ immunoreactive habenular efferents coursed throughout the DV extent of the

Fig. 3. Lov and Ron distribution reflects directional asymmetry of habenulae. (A,C,E) Approximately 50% of Roep larvae show directional asymmetry of the epithalamus similar to WT (situs solitus), with a left-positioned parapineal, expanded lov (n=17/41) and reduced ron (n=13/24) and dex (n=10/25) expression in the left habenula. (B,D,F) Remaining Roep larvae show mirror image reversal (situs inversus) in parapineal position and habenular gene expression patterns. (A-F) Parapineal position (arrowhead) was confirmed by otx5 expression (Gamse et al., 2002). Dorsal views, anterior to top; scale bar: 50 ␮m. (G,H) Lov protein levels are higher in left habenula and FR of WT larvae (see Fig. 2) and half of Roep larvae (G; n=39/70). The other Roep larvae show a L-R reversal in Lov immunofluorescence (H; n=31/70). (I,J) Ron immunofluorescence is expanded in WT right habenula (see Fig. 2) and in half of Roep larvae (I; n=39/70). In L-R reversed Roep larvae, the Ron+ domain is expanded on the left (J; n=31/70). G-J are confocal Z-stacks from the dorsal aspect, anterior to top; scale bars: 40 ␮m. (K) In Roep larvae with a WT pattern of habenular asymmetry, Lov+ and Ron+ efferents project to dorsoventral and ventral IPN domains, respectively. (L) In L-R reversed larvae, projections along the DV axis of IPN are maintained, although Lov+ and Ron+ efferents emerge from the opposite side of the brain. (K,L) Confocal Z-stacks from the lateral aspect, anterior to left. Asterisk and arrowhead indicate anterior and posterior domains of dorsal IPN. Scale bar: 30 ␮m. Asymmetry of habenular projections 4875

Table 1. Randomization of left-right asymmetry in Southpaw-depleted larvae Pancreas position lov expression pattern % of larvae % Right % Left % Midline Tg(foxD3:GFP) n=300 sinistral* 100† 100 0 0 spaw MO in Tg(foxD3:GFP) n=418 sinistral 52 31 18 3 dextral 45 25 17 3 symmetric 3 1 2 0

*Sinistral refers to the wild-type expression pattern of lov in the habenular nuclei with an expanded domain and higher levels of expression in the left habenula (Gamse et al., 2003). Dextral refers to the opposite pattern found in L-R reversed larvae (see Fig. 3). †Absence or L-R reversal of lov expression was not observed in Tg(foxD3:GFP) control larvae. Only one larva (0.3%) had symmetric expression of lov in the habenulae and a right-sided pancreas.

assaying ron and dex expression in larvae lacking the adult IPN, while Ron+ axons were confined to the ventral parapineal. region (Fig. 6A-C, n=3). This DV pattern of immunolabeled Removal of the parapineal by laser-mediated ablation was habenular projections was unaltered in adults derived from performed at 30 hours, prior to the appearance of lov, ron, and spaw MO-injected embryos (n=6), irrespective of the direction dex transcripts in the habenular region. When assessed at 4 of epithalamic laterality (compare Fig. 6D-F and G-I). days, the left habenula of parapineal-ablated larvae showed reduced lov expression (Gamse et al., 2003) (Fig. 7B) and Habenular asymmetry directs IPN connectivity expanded expression of ron and dex (compare Fig. 7C,D and Prior studies demonstrated that habenular asymmetry is E,F). The expression patterns of all genes appeared bilaterally dependent on the parapineal (Concha et al., 2003; Gamse et al., symmetric, with both habenulae exhibiting transcriptional 2003). Following parapineal ablation, the left habenula fails to subdomains more typical of the right habenula. adopt its characteristic properties, such as a larger size, In parapineal-ablated larvae, Lov protein levels were expanded dense neuropil and increased lov expression relative reduced and Ron+ domains expanded in the left habenula, to the right habenula. We examined whether the parapineal also relative to control-ablated larvae (compare Fig. 7G,H and I,J). influences properties associated with the right habenula by Fewer Lov+ axons projected within the left FR, whose immunolabeling now resembled that of the right FR. In contrast to L-R reversal of parapineal position, ablation of the parapineal disrupted Development the DV distribution of habenular efferents onto the IPN. The two Lov+ domains normally observed at the dorsal IPN of control-ablated larvae

Fig. 4. Selective labeling of L-R habenulae in adult zebrafish brain. (A) Schematic of adult zebrafish dorsal brain. The paired habenular nuclei lie at the dorsal surface of the diencephalon posterior to the telencephalon (Te) and anterior to the optic tectum. The habenular commissure was severed and DiI was injected into the right habenula (red) and DiO into the left habenula (green) (Aizawa et al., 2005). (B) Schematic of adult habenulointerpeduncular tract (sagittal view, anterior left) indicating approximate positions of transverse sections (150 ␮m) in D-L: I, D-F; II, G-I and III, J-L. (C) DiO- and (D-L) DiI-labeled left and right habenular projections along the fasciculus retroflexus to IPN viewed dorsally. Fluorescent images were captured using GFP3 (D,G,J) or rhodamine filter sets (E,H,K). (F,I,L) Digital overlay of DiO and DiI images. Dorsal (d) and ventral (v) regions of the target are indicated in L. Scale bar: 120 ␮m. 4876 Development 132 (21) Research article

Fig. 6. DV difference in Lov+ and Ron+ habenular projections onto Fig. 5. Directional asymmetry influences IPN projection pattern. the adult IPN. Confocal images of anti-Lov (red) and anti-Ron Habenular projections onto the IPN in sections (150 ␮m) through (green) double-labeled horizontal sections through adult midbrain of adult midbrain of (A-C) WT and (D-I) spaw MO-injected fish, which (A-C) WT or (D-F) left-positioned parapineal and (G-I) right- Development as larvae had a left (D-F) or right (G-I) positioned parapineal. positioned parapineal fish selected from spaw MO-injected (A-F) Axons originating from left habenula innervate dorsal and Tg(foxD3:GFP) embryos and reared separately. Vibratome sections ventral IPN domains, while those from the right habenula only (150 ␮m) vary slightly in their coordinates along anteroposterior project ventrally. (G-I) In fish with reversed epithalamic laterality, L- axis of the brain, accounting for differences in immunofluorescent R origin of IPN projections is also reversed, such that axons from left signals. Although intensity of labeling varies between given habenula now innervate only the ventral IPN. A-C are higher sections, DV regionalization of immunoreactive habenular axons at magnification Leica MZFLIII images of the same brain as in Fig. 4. the IPN is highly stereotypic. Dorsal is at the top in all images. D,E and G,H were imaged using a Leica SP2 confocal microscope Scale bar: 60 ␮m. and merged to produce the composite images in F,I. Dorsal is at the top in all images. Scale bar: 60 ␮m. left habenula. Here, we show that left and right habenulae display asymmetric patterns of expression for three related (asterisk and arrowhead, Fig. 7K) were reduced to one small genes, lov, ron and dex, which allows habenular subdomains anterior domain (asterisk in Fig. 7L) and an increase in to be distinguished molecularly in the larval brain. Antibodies immunofluorescence was detected at the ventral IPN (Fig. 7L). directed against Lov and Ron proteins further define the These results are consistent with left habenular efferents trajectories and specificity of efferents from the L-R habenulae adopting an IPN projection pattern more characteristic of those and affirm the existence of asymmetry at the level of protein derived from the right habenula. distribution. Reversal of parapineal position is correlated with a mirror image reversal of habenular subdomains and of the molecular specificity of habenular efferents that course through Discussion the bilateral FR. Loss of the parapineal causes the left habenula Inroads into the understanding of the developmental and to develop properties more similar to the right habenula. genetic basis of brain asymmetry have come from the zebrafish Ultimately, this alteration in diencephalic asymmetry has system. Previous work revealed that epithalamic laterality consequences on habenular connectivity at the midbrain target. arises in a step-wise manner and that the direction of asymmetry is determined by Nodal signaling in the early leftover-related gene expression defines habenular embryo (Concha et al., 2000; Liang et al., 2000; Long et al., subdomains 2003; Rebagliati et al., 1998b; Sampath et al., 1998). In the Asymmetric specialization of the habenular nuclei has been vast majority of larvae, left-sided positioning of the parapineal studied extensively in amphibians (see Concha and Wilson, impacts on the differentiation of an adjacent brain nucleus, the 2001). In species of Rana, the right dorsal habenula consists Asymmetry of habenular projections 4877

Fig. 7. The parapineal mediates asymmetry of the habenulointerpeduncular system. (A,C,E) Control-ablated larvae (dashed brackets indicate anteroposterior extent of gene expression). otx5 expression confirmed parapineal position (arrowhead in A,C,E) and extent of ablation. (B,D,F) Parapineal ablation results in reduced lov (B; n=24), and expanded ron (D; n=58) and dex (F; n=16) expression in left habenula (4 days). A-F are dorsal views, anterior to top; scale bar: 50 ␮m. (G-J) Control-ablated Tg(foxd3:GFP) larvae (n=27) show WT pattern of Lov (G) and Ron (I) immunofluorescence. GFP is detected in the pineal (dotted oval) and parapineal (arrowhead) of transgenic larvae even after fixation and antibody labeling. Following parapineal ablation (n=42), Lov labeling (H) was reduced in the left habenula and axons in the left FR, while Ron immunofluorescence (J) expanded in the left habenula. G-J are confocal Z-stacks from the dorsal aspect, anterior to top; scale bars: 40 ␮m for G,H and I,J. (K) Control ablated larvae show the same IPN projection pattern as WT (see Fig. 2). (L) In parapineal ablated larvae, Lov+ projections to dorsal IPN are reduced and Ron+ axons increased ventrally. Asterisk and arrowhead in K indicate anterior and posterior domains of dorsal IPN. K,L are confocal Z-stacks from the lateral aspect anterior to the left; scale bar: 30 ␮m.

basis of biochemical and subcellular properties. For example, only the more lateral region of the left medial habenula shows intense nitric oxide synthase activity (Guglielmotti and Fiorino, 1999). Evidence for habenular subnuclear specialization has also come from studies on cartilaginous and teleost fish. Silver staining of the adult red stingray brain enabled the identification of discrete asymmetric subdomains with differences in neuronal size, shape and density of packing (Iwahori et al., 1991a; Iwahori et al., 1991b). The left medial habenula of salmon exhibits a serotoninergic subregion that is Development not observed on the right (Ekstrom and Ebbesson, 1988). In mammals, the medial habenulae are the equivalent of the amphibian dorsal habenulae (Concha and Wilson, 2001; Harris et al., 1996). In the adult rat, they consist of five discrete subdomains defined by morphological criteria, including cell packing density, neuronal shape and size, cytoplasmic volume, synaptic vesicles and degree of myelination (Andres et al., 1999). Habenular subdomains possess different neurochemical properties and project to different regions of the IPN, as evidenced by a cholinergic neuronal cluster in the ventral portion of the rat medial habenula that is distinct from dorsally situated Substance P containing neurons (Contestabile et al., 1987; Cuello et al., 1978). In contrast to lower vertebrates, these anatomical and neurochemical subdomains appear symmetric. We also did not observe prominent L-R differences in rats or mice for the habenular-specific expression of the Kctd8 or Kctd12/pfet genes. However, habenular patterns of expression for other mammalian KCTD family members have not yet been determined, nor can we rule out the presence of subtle L-R differences. In the present study, we have shown that the zebrafish lov- related gene family includes three members, lov, ron and dex that are expressed differently by the left and right habenulae. By comparing their patterns, six asymmetric subdomains could be assigned to the left and right habenulae (designated i-vi in of a single nucleus, while the left habenula is composed of a Fig. 8A). One distinctive feature is that the left habenula medial and a lateral subnucleus (Braitenberg and Kemali, exhibits DV compartmentalization in gene expression not 1970; Morgan et al., 1973). The medial subnucleus can be observed for the right. Subdivision of the right habenula is further subdivided into medial and lateral components, on the largely along the anteroposterior axis. DV regionalization of 4878 Development 132 (21) Research article

the left habenula has been noted in other lower vertebrates. A 50% amino acid identity between zebrafish Lov and lateral subnucleus occupies most of the dorsal left habenula in Drosophila Shaker (Gamse et al., 2003)], these proteins might the stingray brain, but is restricted to a smaller medial posterior interact and thereby modulate channel assembly or activity. region on the right (Iwahori et al., 1991a; Iwahori et al., Such a role was recently proposed for KCNRG (K+ channel 1991b). There is also a dorsoventral difference in the regulator encoding gene), which encodes a protein structurally distribution of the calcium-binding protein calretinin A in the similar to Lov-related proteins (Ivanov et al., 2003). Another left habenula of Rana esculenta (Guglielmotti et al., 2004). The potential function is in modulating neuronal responses to significance of asymmetry and the relationship of gene secreted signals from other cells. KCTD11/REN represses expression subdomains with afferent input, habenular signaling by the secreted protein Hedgehog by preventing the ultrastructure, and connectivity are important issues to pursue. downstream transcription factor GLI from entering the nucleus For example, it is known that the parapineal selectively (Di Marcotullio et al., 2004). Two-hybrid library screening, innervates the left habenula in several fish species (see Concha immunoprecipitation and MO depletion should shed more light and Wilson, 2001) and may connect with neurons within a on the cellular functions of zebrafish Lov-related proteins. specific subdomain. Determining the purpose of molecular specialization of the L-R differences in zebrafish habenular projections zebrafish habenulae will require a greater understanding of the The trajectory of habenular efferents along the FR and functions of Lov-related proteins. The conserved N-terminal connection between the habenular nuclei and IPN has been T1 domain, a protein-protein interaction motif that promotes well described and is a highly conserved feature of the oligomerization (Collins et al., 2001), could provide useful vertebrate brain (Butler and Hodos, 1996; Cajal, 1966; clues. In the Shaker family of voltage-gated potassium Sutherland, 1982). Several studies have traced habenular channels, it is required for tetramerization of alpha subunits efferents in fish, notably in the goldfish and trout (Villani et al., into a functional channel and for axonal localization (Gu et al., 1996; Yanez and Anadon, 1996), but the presence of 2003; Li et al., 1992). Preliminary data from yeast two-hybrid topographic DV projections onto the IPN was not previously assays suggest that zebrafish Lov and Ron proteins form appreciated. Axonal tracing did reveal that neurons within homophilic and heterophilic dimers, dependent on the presence particular habenular subdomains innervated specific of the T1 domain (J.T.G., unpublished observations). Since the subregions of the IPN (e.g. Herkenhaum and Nauta, 1979; T1 domain of Lov-related KCTD proteins is conserved with Shibata et al., 1986; Villani et al., 1994), although these were that of voltage-gated K+ channel subunits [e.g. approximately presumed to be bilaterally symmetric projections. Preferential Development

Fig. 8. Schematic of habenular domains and efferent projections in larval zebrafish. (A) Six asymmetric domains in L-R habenular nuclei of 4-day larvae are defined by lov, ron and dex expression. The left habenula is divided into one dorsal and three ventral domains, while four expression domains are organized along the anterior- posterior axis of the right habenula. (B) Normally, left habenular Lov+ Ron– neurons (domain i) project to the dorsal IPN. Ron+ efferents from both habenulae (domains ii, iii, v and vi) project to the ventral IPN. (C) In larvae with reversed epithalamic laterality, FR projections are L-R reversed, but DV innervation of the IPN is unaffected. (D) In parapineal-ablated larvae, the left habenula adopts molecular properties characteristic of the right habenula. The dorsal IPN shows a partial loss of Lov+ axons, while the ventral IPN shows increased immunoreactivity. Remaining Lov+ axons at the dorsal IPN may represent an incomplete transformation of the left habenula to right habenular fate. Asymmetry of habenular projections 4879

innervation of the intermediate and central subregions of the lov, a molecular profile more characteristic of the right IPN by a subset of habenular neurons was observed in the adult habenula. Thus, the parapineal normally functions not only to zebrafish brain (Tomizawa et al., 2001); however, differential promote the acquisition of left-specific gene expression left and right projection patterns were not described. (Gamse et al., 2003), but to repress right-specific gene The discovery of molecular asymmetry in the zebrafish expression in the adjacent left habenula. habenulae (Gamse et al., 2003) suggested that L-R differences Loss of the parapineal also affects habenular connections. could extend to habenular efferents and influence target Lov+ axons innervating the dorsal IPN were reduced while Ron recognition. By selective labeling with lipophilic dyes, we immunoreactivity increased in the ventral IPN, consistent with corroborated the recent findings of Aizawa et al. (Aizawa et left habenular neurons adopting the projection pattern of right al., 2005) that the left and right habenular axons project along habenular neurons (Fig. 8D). However, this transformation different DV extents of the IPN. Those authors concluded that may not be complete because some Lov+ projections persisted there is a “topographic projection of left-sided habenular axons in the anterior region of the dorsal IPN. Dye labeling of larval to the dorsal region of the IPN and of the right-sided habenular or adult brains derived from parapineal-ablated embryos axons to the ventral IPN.” While our data support the should resolve the L-R origin of the Lov+ axonal endings that projection of axons from the L and R medial habenulae to remain at the dorsal IPN. topographically different domains along the DV axis of the Parapineal ablation causes a local perturbation of zebrafish IPN, we find that there is substantial overlap in L and diencephalic asymmetry that is not expected to affect the R projections within the ventral region. Thus, the assertion that properties of the midbrain target directly, such as the left and right information is laterotopically represented onto expression of attractive or repulsive guidance cues. Therefore, discrete dorsal and ventral regions of the target nucleus we conclude that laterality of the habenular nuclei influences (Aizawa et al., 2005) is an oversimplification of the pattern of IPN connectivity by altering the molecular properties of connectivity documented in our experiments. habenular axons and presumably their choice of which target Furthermore, the finding that left habenular neurons project subdomains to innervate. How efferents from the right along the entire DV extent of the IPN and right habenular habenula are targeted to only the ventral IPN domain avoiding neurons project predominantly to the ventral IPN is strongly the dorsal one, while left habenula efferents innervate both, is supported by their molecular specificity. Anti-Lov and anti- a problem that will require more information about the types Ron sera label different DV domains of the IPN, domains that and distribution of axon guidance molecules at the IPN and closely resemble the pattern of left and right habenular neighboring brain regions. Members of the neuropilin gene projections, respectively. Lov+ efferents are found throughout family and the netrin 1 receptor Dcc (Deleted in colorectal the DV extent of the IPN, while Ron+ efferents are confined to cancer) are strongly expressed in the habenular nuclei of mouse the ventral region. embryos (Funato et al., 2000; Shu et al., 2000). Repulsive Semaphorin 3F (Sema3F) and attractive Netrin 1 signals have Development Directional asymmetry of the epithalamus been implicated in directing the ventroposterior outgrowth of influences target recognition habenular efferents along the diencephalic neuromere Our results support a role for Nodal signaling in setting the boundary (Funato et al., 2000). Moreover, mutations of direction of asymmetry in the zebrafish epithalamus that neuropilin 2 or Sema3F lead to defasciculation of the FR extends to epithalamic projections. In Roep larvae, the ability (Giger et al., 2000; Sahay et al., 2003). Although the studies to respond to Nodal signaling is partially restored but is in mice provide information about FR navigation toward the insufficient to direct brain laterality. Therefore, at a population midbrain, little is known about the nature of guidance cues level, the direction of brain asymmetry is L-R randomized. habenular axons receive at the IPN. With the aid of GFP However, even in individuals that show a mirror image reversal transgenes that highlight habenular projections (Parinov et al., of habenular asymmetry and in the L-R pattern of 2004), forward genetic screens in zebrafish may identify such immunoreactive Lov+ and Ron+ habenular efferents, DV cues. connections onto the IPN appear unaffected (Fig. 8C). Parallel The demonstration that directional asymmetry in one region studies on adult zebrafish that lacked the Nodal signal of the brain guides connectivity in a distant region has Southpaw as embryos also indicate that reversal of habenular interesting implications for deciphering the origin of laterality asymmetry changes the L-R origin of inputs to the IPN, but not in the mammalian cortex. Further exploration into the DV innervation of the IPN. Therefore, development of the generation of L-R connectivity differences could also prove midbrain target and its putative DV guidance cues appear relevant for understanding human neurological disorders that intact, suggesting that the IPN is not directly modified by have been associated with abnormal neuroanatomical Nodal signals. asymmetry (Bruder, 2003; Green et al., 2003).

Habenular asymmetry determines the dorsoventral We thank Michael Rebagliati for southpaw reagents and Ona pattern of IPN connectivity Martin and Yixian Zheng for expert guidance on protein purification In contrast to genetically altered larvae, where global or early- and antibody preparation. We are grateful to Chen-Ming Fan, Joseph acting effects of Nodal activity on the developing neural tube Gall, Francesco Argenton and Lucilla Facchin for valuable discussions, and Lea Fortuno and Michael Willey for technical help. cannot be completely ruled out, selective ablation of the Support was provided by an American Cancer Society post-doctoral parapineal provides a rigorous test of the correlation between fellowship (J.T.G.), funds from the Institut National de la Santé et de habenular laterality and IPN connectivity. la Recherche Médicale, the Centre National de la Recherche After parapineal ablation, the left habenula showed Scientifique, the Hôpital Universitaire de Strasbourg, the Association increased expression of ron and dex and reduced expression of pour la Recherche sur le Cancer and the Ligue Nationale Contre le 4880 Development 132 (21) Research article

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