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