Marnie E. Halpern

Genes dependent on zebrafish cyclops function identified by AFLP differential gene expression screen.

Summary: Zebrafish cyclops (cyc) encodes a Transforming Growth Factor β (TGFβ) signaling factor closely related to mouse Nodal. By comparing amplified fragment length polymorphisms (AFLP) from cyc mutant and wild‐type cDNA pools, we devised a differential gene expression screen to isolate genes whose expression is dependent on Cyc signaling. We report two genes not previously described in the zebrafish that were identified using this approach. The first gene, crestin, is expressed predominantly in premigratory and migrating neural crest cells during somitogenesis stages. crestin expression is reduced in cyc mutants initially but recovers by late somitogenesis. The second gene encodes the zebrafish homologue of the calcium‐binding protein, calreticulin. Zebrafish calreticulin is highly expressed in the hatching gland and in the floor plate, tissues that are affected in cyc mutants. During gastrulation, calreticulin transcripts are found in the dorsal mesendoderm, in the same cells that express the cyc gene. Expression is reduced in cyc mutants and is abolished by the one‐eyed pinhead (oep) mutation that is presumed to prevent Nodal signaling. The identification of calreticulin suggests that a differential screen between wild‐type and mutant cDNA is a useful approach to reveal regulation of unexpected gene expression in response to cellular signals. genesis 26:86–97, 2000.

Unexpected diversity and photoperiod dependence of the zebrafish melanopsin system.

Animals have evolved specialized photoreceptors in the retina and in extraocular tissues that allow them to measure light changes in their environment. In mammals, the retina is the only structure that detects light and relays this information to the brain. The classical photoreceptors, rods and cones, are responsible for vision through activation of rhodopsin and cone opsins. Melanopsin, another photopigment first discovered in Xenopus melanophores (Opn4x), is expressed in a small subset of retinal ganglion cells (RGCs) in the mammalian retina, where it mediates non-image forming functions such as circadian photoentrainment and sleep. While mammals have a single melanopsin gene (opn4), zebrafish show remarkable diversity with two opn4x-related and three opn4-related genes expressed in distinct patterns in multiple neuronal cell types of the developing retina, including bipolar interneurons. The intronless opn4.1 gene is transcribed in photoreceptors as well as in horizontal cells and produces functional photopigment. Four genes are also expressed in the zebrafish embryonic brain, but not in the photoreceptive pineal gland. We discovered that photoperiod length influences expression of two of the opn4-related genes in retinal layers involved in signaling light information to RGCs. Moreover, both genes are expressed in a robust diurnal rhythm but with different phases in relation to the light-dark cycle. The results suggest that melanopsin has an expanded role in modulating the retinal circuitry of fish.

Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway

Significance The forebrain habenular nuclei (Hb) and their connections to the midbrain interpeduncular nucleus (IPN) have emerged as a valuable model to study left-right differences in the zebrafish brain. However, whether this pathway is enriched in the neurotransmitter acetylcholine and involved in nicotine addiction as in mammals is unresolved. We discovered a duplicated cholinergic gene locus that is predominantly expressed in the right Hb at larval stages. Through electrophysiology and pharmacology, we show that this asymmetrical cholinergic pathway is functional. Moreover, specific nicotinic acetylcholine receptor subunits localize to the same subregions of the IPN that are activated by exposure of adults to nicotine. Our study firmly establishes the zebrafish as a valid model to study how Hb-IPN circuitry influences nicotine addiction. The habenulo-interpeduncular pathway, a highly conserved cholinergic system, has emerged as a valuable model to study left-right asymmetry in the brain. In larval zebrafish, the bilaterally paired dorsal habenular nuclei (dHb) exhibit prominent left-right differences in their organization, gene expression, and connectivity, but their cholinergic nature was unclear. Through the discovery of a duplicated cholinergic gene locus, we now show that choline acetyltransferase and vesicular acetylcholine transporter homologs are preferentially expressed in the right dHb of larval zebrafish. Genes encoding the nicotinic acetylcholine receptor subunits α2 and β4 are transcribed in the target interpeduncular nucleus (IPN), suggesting that the asymmetrical cholinergic pathway is functional. To confirm this, we activated channelrhodopsin-2 specifically in the larval dHb and performed whole-cell patch-clamp recording of IPN neurons. The response to optogenetic or electrical stimulation of the right dHb consisted of an initial fast glutamatergic excitatory postsynaptic current followed by a slow-rising cholinergic current. In adult zebrafish, the dHb are divided into discrete cholinergic and peptidergic subnuclei that differ in size between the left and right sides of the brain. After exposing adults to nicotine, fos expression was activated in subregions of the IPN enriched for specific nicotinic acetylcholine receptor subunits. Our studies of the newly identified cholinergic gene locus resolve the neurotransmitter identity of the zebrafish habenular nuclei and reveal functional asymmetry in a major cholinergic neuromodulatory pathway of the vertebrate brain.

Neurotransmitter map of the asymmetric dorsal habenular nuclei of zebrafish

The role of the habenular nuclei in modulating fear and reward pathways has sparked a renewed interest in this conserved forebrain region. The bilaterally paired habenular nuclei, each consisting of a medial/dorsal and lateral/ventral nucleus, can be further divided into discrete subdomains whose neuronal populations, precise connectivity, and specific functions are not well understood. An added complexity is that the left and right habenulae show pronounced morphological differences in many non‐mammalian species. Notably, the dorsal habenulae of larval zebrafish provide a vertebrate genetic model to probe the development and functional significance of brain asymmetry. Previous reports have described a number of genes that are expressed in the zebrafish habenulae, either in bilaterally symmetric patterns or more extensively on one side of the brain than the other. The goal of our study was to generate a comprehensive map of the zebrafish dorsal habenular nuclei, by delineating the relationship between gene expression domains, comparing the extent of left–right asymmetry at larval and adult stages, and identifying potentially functional subnuclear regions as defined by neurotransmitter phenotype. Although many aspects of habenular organization appear conserved with rodents, the zebrafish habenulae also possess unique properties that may underlie lateralization of their functions. genesis 52:636–655, 2014.

Disruption of Epithalamic Left–Right Asymmetry Increases Anxiety in Zebrafish

Differences between the left and right sides of the brain are found throughout the animal kingdom, but the consequences of altered neural asymmetry are not well understood. In the zebrafish epithalamus, the parapineal is located on the left side of the brain where it influences development of the adjacent dorsal habenular (dHb) nucleus, causing the left and right dHb to differ in their organization, gene expression, and connectivity. Left–right (L-R) reversal of parapineal position and dHb asymmetry occurs spontaneously in a small percentage of the population, whereas the dHb develop symmetrically following experimental ablation of the parapineal. The habenular region was previously implicated in modulating fear in both mice and zebrafish, but the relevance of its L-R asymmetry is unclear. We now demonstrate that disrupting directionality of the zebrafish epithalamus causes reduced exploratory behavior and increased cortisol levels, indicative of enhanced anxiety. Accordingly, exposure to buspirone, an anxiolytic agent, significantly suppresses atypical behavior. Axonal projections from the parapineal to the dHb are more variable when it is located on the right side of the brain, revealing that L-R reversals do not necessarily represent a neuroanatomical mirror image. The results highlight the importance of directional asymmetry of the epithalamus in the regulation of stress responses in zebrafish. SIGNIFICANCE STATEMENT The asymmetric epithalamus of zebrafish has emerged as a valuable model to explore the formation and function of left–right differences in the brain. To probe the relationship between brain laterality and behavior, we examined the effects of left–right reversal of epithalamic asymmetry or symmetric development on behavior. In both cases, zebrafish showed increased measures of fear/anxiety, including reduced exploratory behavior and delayed exit from a confined space. Adults with reversed L-R asymmetry also have elevated cortisol levels relative to controls. The results reveal the importance of directional asymmetry of the dorsal diencephalon in the modulation of anxiety.

Aversive cues fail to activate fos expression in the asymmetric olfactory-habenula pathway of zebrafish

The dorsal habenular nuclei of the zebrafish epithalamus have become a valuable model for studying the development of left-right (L-R) asymmetry and its function in the vertebrate brain. The bilaterally paired dorsal habenulae exhibit striking differences in size, neuroanatomical organization, and molecular properties. They also display differences in their efferent connections with the interpeduncular nucleus (IPN) and in their afferent input, with a subset of mitral cells distributed on both sides of the olfactory bulb innervating only the right habenula. Previous studies have implicated the dorsal habenulae in modulating fear/anxiety responses in juvenile and adult zebrafish. It has been suggested that the asymmetric olfactory-habenula pathway (OB-Ha), revealed by selective labeling from an lhx2a:YFP transgene, mediates fear behaviors elicited by alarm pheromone. Here we show that expression of the fam84b gene demarcates a unique region of the right habenula that is the site of innervation by lhx2a:YFP-labeled olfactory axons. Upon ablation of the parapineal, which normally promotes left habenular identity; the fam84b domain is present in both dorsal habenulae and lhx2a:YFP-labeled olfactory bulb neurons form synapses on the left and the right side. To explore the relevance of the asymmetric olfactory projection and how it might influence habenular function, we tested activation of this pathway using odorants known to evoke behaviors. We find that alarm substance or other aversive odors, and attractive cues, activate fos expression in subsets of cells in the olfactory bulb but not in the lhx2a:YFP expressing population. Moreover, neither alarm pheromone nor chondroitin sulfate elicited fos activation in the dorsal habenulae. The results indicate that L-R asymmetry of the epithalamus sets the directionality of olfactory innervation, however, the lhx2a:YFP OB-Ha pathway does not appear to mediate fear responses to aversive odorants.

Distinct requirements for Wntless in habenular development.

Secreted Wnt proteins play pivotal roles in development, including regulation of cell proliferation, differentiation, progenitor maintenance and tissue patterning. The transmembrane protein Wntless (Wls) is necessary for secretion of most Wnts and essential for effective Wnt signaling. During a mutagenesis screen to identify genes important for development of the habenular nuclei in the dorsal forebrain, we isolated a mutation in the sole wls gene of zebrafish and confirmed its identity with a second, independent allele. Early embryonic development appears normal in homozygous wls mutants, but they later lack the ventral habenular nuclei, form smaller dorsal habenulae and otic vesicles, have truncated jaw and fin cartilages and lack swim bladders. Activation of a reporter for β-catenin-dependent transcription is decreased in wls mutants, indicative of impaired signaling by the canonical Wnt pathway, and expression of Wnt-responsive genes is reduced in the dorsal diencephalon. Wnt signaling was previously implicated in patterning of the zebrafish brain and in the generation of left-right (L-R) differences between the bilaterally paired dorsal habenular nuclei. Outside of the epithalamic region, development of the brain is largely normal in wls mutants and, despite their reduced size, the dorsal habenulae retain L-R asymmetry. We find that homozygous wls mutants show a reduction in two cell populations that contribute to the presumptive dorsal habenulae. The results support distinct temporal requirements for Wls in habenular development and reveal a new role for Wnt signaling in the regulation of dorsal habenular progenitors.

Left-right asymmetry: Advances and enigmas

The coiling of a snail shell, the transformation of an initially simple mesodermal tube into the multichambered heart, or the lateralized sensing of environmental cues by bilaterally paired neurons, are all examples of how left-right (L-R) differences can profoundly affect development, physiology and behavior. Developmental biologists have long been fascinated by the prominent internal asymmetries of outwardly symmetric animals, a fascination piqued in the mid-1990’s by the discovery of the transient, unilateral expression of the Nodal signaling molecule in the left lateral plate mesoderm (LPM) (Levin et al., 1995). This discovery quickly opened the door for the dissection of the genetic pathways that distinguish cells on the left side of an organism from those on its right side. Almost 20 years later, significant progress has been made towards understanding how symmetry is broken, and how L-R biases that are initially small are interpreted and propagated, ultimately triggering dramatic molecular and morphological changes. Alongside these advances, however, a number of processes still remain quite mysterious, and some provoke outright controversy. In part, the difficulties in interpretation come from the fact that the generation of anatomical L-R asymmetry is intimately connected to the development of the other body axes [anterior-posterior (A-P), dorsal-ventral (D-V)] and of specific structures. For example, deficiencies in initiating or propagating differential L-R behavior of cells or tissues can arise with what seem to be relatively minor perturbations of the integrity or timely formation of axial midline structures, which are important influences in maintaining L–R biases. The impetus for this special issue of genesis, The Journal of Genetics and Development came from a satellite symposium held in 2013, in conjunction with the 72 Annual Meeting of the Society for Developmental Biology and the 17 International Congress of Developmental Biology (refer to the meeting review by Burdine and Caspary, 2013). An important goal of the symposium was to bring together researchers working on invertebrate and vertebrate systems and to tackle the problem at multiple levels: from determining how the L-R axis is first established to the asymmetric processes that underlie cellular diversification, organ morphology, and even sophisticated animal behaviors. Disruptions in these processes can lead to dire consequences on human development and health. The collection of reviews and research papers provided in this issue represents some of the topics covered in the symposium and aims to place what has been learned in the context of problems that are still unresolved. Probing how L-R asymmetry arose in evolution may help in deciphering the fundamental mechanisms responsible for breaking symmetry. As outlined by Namigai et al., (458–470), neither of the two leading contenders for vertebrates, the nodal flow hypothesis and the ion-flow hypothesis, adequately explains how L-R differences are established for all bilaterian phyla, despite arguments to the contrary (e.g., Blum et al. 2014; Vandenberg and Levin, 2013). Increasing evidence, such as from studies on the sea urchin (reviewed by Warner and McClay, 481–487) points to an ancestral role for unilateral Nodal signaling in Deuterostomes. However, in invertebrates without obvious nodal gene homologues, chirality is likely accomplished by polarized cytoskeletal elements (and see Vandenburg et al., 2013). A pivotal role for the cytoskeleton is supported by the finding that Myosin ID (MyoID), an actinassociated motor protein, is required for the dextrally directed morphogenesis of the viscera in Drosophila (refer to G eminard et al., 471–480). In C. elegans embryos, a chiral cortical network present at the 1-cell stage is predicted to drive a dextral rotation within the eggshell (Schonegg et al., 572–580). This rotation around the A-P axis later correlates with the skewed formation of the mitotic spindle during the second cleavage division, indicating that the nematode zygote has an intrinsic and invariant chirality that is present earlier than previously thought. However, despite invariable early events, the recovery of spontaneous, temperaturedependent L-R reversals in the asymmetric positioning of the gut and gonad in males of some C. elegans strains (Callander et al., 581–587) suggests that later

Development and connectivity of the habenular nuclei

Accumulating evidence has reinforced that the habenular region of the vertebrate dorsal forebrain is an essential integrating center, and a region strongly implicated in neurological disorders and addiction. Despite the important and diverse neuromodulatory roles the habenular nuclei play, their development has been understudied. The emphasis of this review is on the dorsal habenular nuclei of zebrafish, homologous to the medial nuclei of mammals, as recent work has revealed new information about the signaling pathways that regulate their formation. Additionally, the zebrafish dorsal habenulae have become a valuable model for probing how left-right differences are established in a vertebrate brain. Sonic hedgehog, fibroblast growth factors and Wingless-INT proteins are all involved in the generation of progenitor cells and ultimately, along with Notch signaling, influence habenular neurogenesis and left-right asymmetry. Intriguingly, a genetic network has emerged that leads to the differentiation of dorsal habenular neurons and, through localized chemokine signaling, directs the posterior outgrowth of their newly emerging axons towards their postsynaptic target, the midbrain interpeduncular nucleus.

Convergence of signaling pathways underlying habenular formation and axonal outgrowth in zebrafish

The habenular nuclei are a conserved integrating center in the vertebrate epithalamus, where they modulate diverse behaviors. Despite their importance, our understanding of habenular development is incomplete. Time-lapse imaging and fate mapping demonstrate that the dorsal habenulae (dHb) of zebrafish are derived from dbx1b-expressing (dbx1b+) progenitors, which transition into cxcr4b-expressing neuronal precursors. The precursors give rise to differentiated neurons, the axons of which innervate the midbrain interpeduncular nucleus (IPN). Formation of the dbx1b+ progenitor population relies on the activity of the Shh, Wnt and Fgf signaling pathways. Wnt and Fgf function additively to generate dHb progenitors. Surprisingly, Wnt signaling also negatively regulates fgf8a, confining expression to a discrete dorsal diencephalic domain. Moreover, the Wnt and Fgf pathways have opposing roles in transcriptional regulation of components of the Cxcr4-chemokine signaling pathway. The chemokine pathway, in turn, directs the posterior outgrowth of dHb efferents toward the IPN and, when disrupted, results in ectopic, anteriorly directed axonal projections. The results define a signaling network underlying the generation of dHb neurons and connectivity with their midbrain target. Summary: Development of dHb neurons and posteriorly directed outgrowth of their axons are regulated by Shh, Wnt, Fgf, and chemokine signaling pathways in zebrafish.