Hideomi Tanaka

N-cadherin mediates retinal lamination, maintenance of forebrain compartments and patterning of retinal neurites

The complex, yet highly ordered and predictable, structure of the neural retina is one of the most conserved features of the vertebrate central nervous system. In all vertebrate classes, retinal neurons are organized into laminae with each neuronal class adopting specific morphologies and patterns of connectivity. Using genetic analyses in zebrafish, we demonstrate that N-cadherin (Ncad) has several distinct and crucial functions during the establishment of retinal organization. Although the location of cell division is disorganized in embryos with reduced or no Ncad function, different classes of retinal neurons are generated. However, these neurons fail to organize into correct laminae, most probably owing to compromised adhesion between retinal cells. In addition, amacrine cells exhibit exuberant and misdirected outgrowth of neurites that contributes to severe disorganization of the inner plexiform layer. Retinal ganglion cells also exhibit defects in process outgrowth, with axons exhibiting fasciculation defects and adopting incorrect ipsilateral trajectories. At least some of these defects are likely to be due to a failure to maintain compartment boundaries between eye, optic nerve and brain. Although in vitro studies have implicated Fgf receptors in modulating the axon outgrowth promoting properties of Ncad, most aspects of the Ncad mutant phenotype are not phenocopied by treatments that block Fgf receptor function.

Dual roles of zygotic and maternal Scribble1 in neural migration and convergent extension movements in zebrafish embryos

In the developing vertebrate hindbrain, the characteristic trajectory of the facial (nVII) motor nerve is generated by caudal migration of the nVII motor neurons. The nVII motor neurons originate in rhombomere (r) 4, and migrate caudally into r6 to form the facial motor nucleus. In this study, using a transgenic zebrafish line that expresses green fluorescent protein (GFP) in the cranial motor neurons, we isolated two novel mutants, designated landlocked (llk) and off-road (ord), which both show highly specific defects in the caudal migration of the nVII motor neurons. We show that the landlocked locus contains the gene scribble1 (scrb1), and that its zygotic expression is required for migration of the nVII motor neurons mainly in a non cell-autonomous manner. Taking advantage of the viability of the llk mutant embryos, we found that maternal expression of scrb1 is required for convergent extension (CE) movements during gastrulation. Furthermore, we show a genetic interaction between scrb1 and trilobite(tri)/strabismus(stbm) in CE. The dual roles of the scrb1 gene in both neuronal migration and CE provide a novel insight into the underlying mechanisms of cell movement in vertebrate development.

Frizzled3a and Celsr2 function in the neuroepithelium to regulate migration of facial motor neurons in the developing zebrafish hindbrain

Migration of neurons from their birthplace to their final target area is a crucial step in brain development. Here, we show that expression of the off-limits/frizzled3a (olt/fz3a) and off-road/celsr2 (ord/celsr2) genes in neuroepithelial cells maintains the facial (nVII) motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. In the absence of olt/fz3a expression in the neuroepithelium, nVII motor neurons extended aberrant radial processes towards the ventricular surface and mismigrated radially to the dorsomedial part of the hindbrain. Our findings reveal a novel role for these genes, distinctive from their already known functions, in the regulation of the planar cell polarity (i.e. preventing integration of differentiated neurons into the neuroepithelial layer). This contrasts markedly with their reported role in reintegration of neuroepithelial daughter cells into the neuroepithelial layer after cell division.

The Zebrafish-Secreted Matrix Protein You/Scube2 Is Implicated in Long-Range Regulation of Hedgehog Signaling

The Hedgehog (Hh) signal plays a pivotal role in induction of ventral neuronal and muscle cell types around the midline during vertebrate development [1]. We report that the gene disrupted in zebrafish you mutants, in which Hh signaling is impaired, encodes the secreted matrix protein Scube2. Consistently, epistasis analyses suggested that Scube2 functions upstream of Hh ligands or through a parallel pathway. In addition, overexpression analyses suggested that Scube2 is an essential, but a permissive, mediator of Hh signaling in zebrafish embryos. Surprisingly, the you gene is expressed in the dorsal neural tube, raising the possibility that Scube2 could indirectly act via a long-range regulator of Hh signaling. The dorsal Bmps have a long-range and opposing influence on Hh signaling [2-5]. We show that neural plate patterning is affected in you mutants in a way that is consistent with the aberrant long-range action of a Bmp-dependent signal. We further show that Bmp activity can be attenuated by the coexpression of Scube2. Our data support the idea that Scube2 can modulate the long-range action of Bmp-dependent signaling in the neural tube and somites.

Characterization of neural stem cells and their progeny in the adult zebrafish optic tectum

In the adult teleost brain, proliferating cells are observed in a broad area, while these cells have a restricted distribution in adult mammalian brains. In the adult teleost optic tectum, most of the proliferating cells are distributed in the caudal margin of the periventricular gray zone (PGZ). We found that the PGZ is largely divided into 3 regions: 1 mitotic region and 2 post-mitotic regions-the superficial and deep layers. These regions are distinguished by the differential expression of several marker genes: pcna, sox2, msi1, elavl3, gfap, fabp7a, and s100beta. Using transgenic zebrafish Tg (gfap:GFP), we found that the deep layer cells specifically express gfap:GFP and have a radial glial morphology. We noted that bromodeoxyuridine (BrdU)-positive cells in the mitotic region did not exhibit glial properties, but maintained neuroepithelial characteristics. Pulse chase experiments with BrdU-positive cells revealed the presence of self-renewing stem cells within the mitotic region. BrdU-positive cells differentiate into glutamatergic or GABAergic neurons and oligodendrocytes in the superficial layer and into radial glial cells in the deep layer. These results demonstrate that the proliferating cells in the PGZ contribute to neuronal and glial lineages to maintain the structure of the optic tectum in adult zebrafish.

Dual Roles of Notch in Regulation of Apically Restricted Mitosis and Apicobasal Polarity of Neuroepithelial Cells

How the mitosis of neuroepithelial stem cells is restricted to the apical ventricular area remains unclear. In zebrafish, the mosaic eyes(rw306) (moe/epb41l5(rw306)) mutation disrupts the interaction between the putative adaptor protein Moe and the apicobasal polarity regulator Crumbs (Crb), and impairs the maintenance of neuroepithelial apicobasal polarity. While Crb interacts directly with Notch and inhibits its activity, Moe reverses this inhibition. In the moe(rw306) hindbrain, Notch activity is significantly reduced, and the number of cells that proliferate basally away from the apical area is increased. Surprisingly, activation of Notch in the moe(rw306) mutant rescues not only the basally localized proliferation but also the aberrant neuroepithelial apicobasal polarity. We present evidence that the Crb⋅Moe complex and Notch play key roles in a positive feedback loop to maintain the apicobasal polarity and the apical-high basal-low gradient of Notch activity in neuroepithelial cells, both of which are essential for their apically restricted mitosis.

Novel mutations affecting axon guidance in zebrafish and a role for plexin signalling in the guidance of trigeminal and facial nerve axons

In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial (VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord.

Neuroepithelial cells require fucosylated glycans to guide the migration of vagus motor neuron progenitors in the developing zebrafish hindbrain.

The molecular mechanisms by which neurons migrate and accumulate to form the neural layers and nuclei remain unclear. The formation of vagus motor nuclei in zebrafish embryos is an ideal model system in which to address this issue because of the transparency of the embryos and the availability of established genetic and molecular biological techniques. To determine the genes required for the formation of the vagus motor nuclei, we performed N-ethyl-N-nitrosourea-based mutant screening using a zebrafish line that expresses green fluorescent protein in the motor neurons. In wild-type embryos, the vagus motor neuron progenitors are born in the ventral ventricular zone, then migrate tangentially in the dorsolateral direction, forming the nuclei. However, in towhead (twdrw685) mutant embryos, the vagus motor neuron progenitors stray medially away from the normal migratory pathway and fail to stop in the right location. The twdrw685 mutant has a defect in the GDP-mannose 4,6 dehydratase (gmds) gene, which encodes a key enzyme in the fucosylation pathway. Levels of fucosylated glycans were markedly and specifically reduced in twdrw685 mutant embryos. Cell transplantation analysis revealed that GMDS is not essential in the vagus motor neuron progenitors for correct formation of the vagus motor nuclei, but is required in the neuroepithelial cells that surround the progenitors. Together, these findings suggest that fucosylated glycans expressed in neuroepithelial cells are required to guide the migration of vagus motor neuron progenitors.

Islet1 selectively promotes peripheral axon outgrowth in Rohon-Beard primary sensory neurons

We isolated a novel zebrafish mutant, lullaby (llb), and showed that the llb locus encodes the zebrafish orthologue of isl1. Rohon‐Beard (RB) primary sensory neurons are multipolar neurons that extend their central axons longitudinally within the spinal cord and also extend their peripheral axons under the skin. In llb embryos, the outgrowth of the peripheral axons of RB neurons was selectively impaired, which correlated with down‐regulation of the expression of dihydropyrimidinase‐like 3 (dpysl3, also known as collapsin response mediator protein 4, crmp4). Antisense morpholino oligonucleotide (AMO)‐mediated knockdown of dpysl3 inhibited the outgrowth of the peripheral axons of RB neurons, and semaphorin 3d (sema3d) AMO enhanced this effect. These data indicate that Dpysl3 is cooperating with Sema3d in the peripheral axon outgrowth, and Isl1 is required for the selective outgrowth of the peripheral axons of RB neurons by maintaining the expression of dpysl3. Developmental Dynamics 240:9–22, 2011.

Dpysl2 (CRMP2) and Dpysl3 (CRMP4) phosphorylation by Cdk5 and DYRK2 is required for proper positioning of Rohon-Beard neurons and neural crest cells during neurulation in zebrafish.

Dpysl2 (CRMP2) and Dpysl3 (CRMP4) are involved in neuronal polarity and axon elongation in cultured neurons. These proteins are expressed in various regions of the developing nervous system, but their roles in vivo are largely unknown. In dpysl2 and dpysl3 double morphants, Rohon-Beard (RB) primary sensory neurons that were originally located bilaterally along the midline shifted their position to a more medial location in the dorsal-most part of spinal cord. A similar phenotype was observed in the cdk5 and dyrk2 double morphants. Dpysl2 and Dpysl3 phosphorylation mimics recovered this phenotype. Cell transplantation analysis demonstrated that this ectopic RB cell positioning was non-cell autonomous and correlated with the abnormal position of neural crest cells (NCCs), which also occupied the dorsal-most part of the spinal cord during the neural rod formation stage. The cell position of other interneuron and motor neurons within the central nervous system was normal in these morphants. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and DYRK2 is required for the proper positioning of RB neurons and NCCs during neurulation in zebrafish embryos.