Bernard Thisse

The parapineal mediates left-right asymmetry in the zebrafish diencephalon

The dorsal diencephalon (or epithalamus) of larval zebrafish displays distinct left-right asymmetries. The pineal complex consists of the pineal organ anlage and an unpaired, left-sided accessory organ – the parapineal. The neighboring brain nuclei, the left and right dorsal habenulae, show consistent differences in their size, density of neuropil and gene expression. Mutational analyses demonstrate a correlation between the left-right position of the parapineal and the laterality of the habenular nuclei. We show that selective ablation of the parapineal organ results in the loss of habenular asymmetry. The left-sided parapineal therefore influences the left-right identity of adjacent brain nuclei, indicating that laterality of the dorsal diencephalon arises in a step-wise fashion.

Otx5 regulates genes that show circadian expression in the zebrafish pineal complex

The photoneuroendocrine system translates environmental light conditions into the circadian production of endocrine and neuroendocrine signals. Central to this process is the pineal organ, which has a conserved role in the cyclical synthesis and release of melatonin to influence sleep patterns and seasonal reproduction. In lower vertebrates, the pineal organ contains photoreceptors whose activity entrains an endogenous circadian clock and regulates transcription in pinealocytes. In mammals, pineal function is influenced by retinal photoreceptors that project to the suprachiasmatic nucleus—the site of the endogenous circadian clock. A multisynaptic pathway then relays information about circadian rhythmicity and photoperiod to the pineal organ. The gene cone rod homeobox (crx), a member of the orthodenticle homeobox (otx) family, is thought to regulate pineal circadian activity. In the mouse, targeted inactivation of Crx causes a reduction in pineal gene expression and attenuated entrainment to light/dark cycles. Here we show that crx and otx5 orthologs are expressed in both the pineal organ and the asymmetrically positioned parapineal of larval zebrafish. Circadian gene expression is unaffected by a reduction in Crx expression but is inhibited specifically by depletion of Otx5. Our results indicate that Otx5 rather than Crx regulates genes that show circadian expression in the zebrafish pineal complex.

Directional asymmetry of the zebrafish epithalamus guides dorsoventral innervation of the midbrain target

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

Chemokine Signaling Guides Axons within the Retina in Zebrafish

Chemokines are a large family of secreted proteins that play an important role in the migration of leukocytes during hematopoiesis and inflammation. Chemokines and their receptors are also widely distributed in the CNS. Although recent investigations are beginning to elucidate chemokine function within the CNS, relatively little is known about the CNS function of this important class of molecules. To better appreciate the CNS function of chemokines, the role of signaling by stromal cell-derived factor-1 (SDF-1) through its receptor, chemokine (CXC motif) receptor 4 (CXCR4), was analyzed in zebrafish embryos. The SDF-1/CXCR4 expression pattern suggested that SDF-1/CXCR4 signaling was important for guiding retinal ganglion cell axons within the retina to the optic stalk to exit the retina. Antisense knockdown of the ligand and/or receptor and a genetic CXCR4 mutation both induced retinal axons to follow aberrant pathways within the retina. Furthermore, retinal axons deviated from their normal pathway and extended to cells ectopically expressing SDF-1 within the retina. These data suggest that chemokine signaling is both necessary and sufficient for directing retinal growth cones within the retina.

A critical role for calponin 2 in vascular development.

Calponin 2 (h2 calponin, CNN2) is an actin-binding protein implicated in cytoskeletal organization. We have found that the expression of calponin 2 is relatively restricted to vasculature from 16 to 30 h post-fertilization during zebrafish (Danio rerio) development. Forty-eight hours after injecting antisense morpholino oligos against calponin 2 into embryos at the 1-4-cell stage, zebrafish demonstrated various cardiovascular defects, including sluggish axial and head circulation, absence of circulation in intersegmental vessels and in the dorsal longitudinal anastomotic vessel, enlarged cerebral ventricles, and pericardial edema, in addition to an excess bending, spiraling tail and twisting of the caudal fin. Knockdown of calponin 2 in the Tg(fli1:EGFP)y1 zebrafish line (in which a fli1 promoter drives vascular-specific enhanced green fluorescent protein expression) indicated that diminished calponin 2 expression blocked the proper migration of endothelial cells during formation of intersegmental vessels. In vitro studies showed that basic fibroblast growth factor-induced human umbilical vein endothelial cell migration was down-regulated by knockdown of calponin 2 expression using an antisense adenovirus, and overexpression of calponin 2 enhanced migration and hastened wound healing. These events were correlated with activation of mitogen-activated protein kinase; moreover, inhibition of this pathway blocked the promigratory effect of calponin 2. Collectively, these data suggest that calponin 2 plays an important role in the migration of endothelial cells both in vivo and in vitro and that its expression is critical for proper vascular development.

Generation of Ectopic Morphogen Gradients in the Zebrafish Blastula

In the zebrafish embryo, cells of the early blastula animal pole are all equivalent and are fully pluripotent until the midblastula transition that occurs at the tenth cell cycle (512 to 1K cells). This naive territory of the embryo is therefore perfectly suited to assay for morphogen activity. Here we describe different methods to generate ectopic morphogen gradients, either in vivo at the animal pole of the embryo, or in vitro in animal pole explants or in aggregates of animal pole blastomeres (also named embryoid bodies). These methods include injection of mRNA coding for growth factor(s) into animal pole blastomere(s), transplantation of growth factor(s) secreting cells, implantation of beads coated with purified protein(s), and various combinations of these different approaches. Our comparative study reveals that all these methods allow to generate morphogen gradient(s) that are able to induce, both in vivo and in vitro, the formation of a well-patterned embryonic axis.