RESUMEN

Rett syndrome is a severe neurodevelopmental disorder. It is caused by a mutation in methyl-CpG binding protein 2 (MecP2), a transcriptional regulator that recruits protein complexes involved in histone modification and chromatin remodeling. However, the role of Mecp2 in Rett syndrome remains unclear. In this study, we investigated the function of Mecp2 in neuronal development using zebrafish embryos. Mecp2 expression was detected ubiquitously in the central nervous system and muscles at 28 h postfertilization (hpf). We injected an antisense morpholino oligonucleotide (AMO) to induce Mecp2 knockdown phenotype. In mecp2 morphants (embryos with Mecp2 knockdown by AMO) at 28 and 72 hpf, we found an increase in abnormal axonal branches of caudal primary motor neurons and a decrease in motor activity. In mecp2 morphants at 24 hpf, we observed an increase in the expression of an mecp2 downstream candidate gene, brain derived neurotrophic factor (bdnf). In mecp2 morphants at 72 hpf, the presynaptic area stained by an anti-SV2 antibody was increased at the neuromuscular junction (NMJ). Interestingly, the size of SV2-positive presynaptic area at the NMJ was also increased following bdnf mRNA injection, while it was normalized in a double knockdown of mecp2 and bdnf. These results imply that Mecp2 is an important functional regulator of bdnf gene expression during neural circuit formation in zebrafish embryo. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1101-1113, 2017.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica/fisiología , Proteína 2 de Unión a Metil-CpG/metabolismo , Neuronas Motoras/citología , Unión Neuromuscular/metabolismo , Proyección Neuronal/fisiología , Factores de Edad , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Diferenciación Celular , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Larva , Proteína 2 de Unión a Metil-CpG/genética , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Neuronas Motoras/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Proyección Neuronal/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Estimulación Física , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , ARN Mensajero/metabolismo , ARN Mensajero/farmacología , Tubulina (Proteína)/metabolismo , Pez Cebra

RESUMEN

Adult neurogenesis attracts broad attention as a possible cure for neurological disorders. However, its regulatory mechanism is still unclear. Therefore, they have been studying the cell proliferation mechanisms of neural stem cells (NSCs) using zebrafish, which have high regenerative potential in the adult brain. The presence of neuroepithelial-type NSCs in the optic tectum of adult zebrafish has been previously reported. In the present study, it was first confirmed that NSCs in the optic tectum decrease or increase in proportion to projection of the optic nerves from the retina. At 4 days after optic nerve crush (ONC), BrdU-positive cells decreased in the optic tectum's operation side. In contrast, at 3 weeks after ONC, BrdU-positive cells increased in the optic tectum's operation side. To study the regulatory mechanisms, they focused on the BDNF/TrkB system as a regulatory factor in the ONC model. It was found that bdnf was mainly expressed in the periventricular gray zone (PGZ) of the optic tectum by using in situ hybridization. Interestingly, expression level of bdnf significantly decreased in the optic tectum at 4 days after ONC, and its expression level tended to increase at 3 weeks after ONC. They conducted rescue experiments using a TrkB agonist and confirmed that decrease of NSC proliferation in the optic tectum by ONC was rescued by TrkB signal activation, suggesting stimuli-dependent regulation of NSC proliferation in the optic tectum of adult zebrafish. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proliferación Celular/fisiología , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Traumatismos del Nervio Óptico , Receptor trkB/metabolismo , Colículos Superiores/fisiología , Proteínas de Pez Cebra/metabolismo , Animales , Modelos Animales de Enfermedad , Células-Madre Neurales/citología , Receptor trkB/agonistas , Colículos Superiores/citología , Pez Cebra

RESUMEN

The zebrafish brain can continue to produce new neurons in widespread neurogenic brain regions throughout life. In contrast, neurogenesis in the adult mammalian brain is restricted to the subventricular zone (SVZ) and dentate gyrus (DG). In neurogenic regions in the adult brain, radial glial cells (RGCs) are considered to function as neural stem cells (NSCs). We generated a Tg(gfap:Gal4FF) transgenic zebrafish line, which enabled us to express specific genes in RGCs. To study the function of RGCs in neurogenesis in the adult zebrafish brain, we also generated a Tg(gfap: Gal4FF; UAS:nfsB-mcherry) transgenic zebrafish line, which allowed us to induce cell death exclusively within RGCs upon addition of metronidazole (Mtz) to the media. RGCs expressing nitroreductase were specifically ablated by the Mtz treatment, decreasing the number of proliferative RGCs. Using the Tg(gfap:Gal4FF; UAS:nfsB-mcherry) transgenic zebrafish line, we found that RGCs were specifically ablated in the adult zebrafish telencephalon. The Tg(gfap:Gal4FF) line could be useful to study the function of RGCs.

Asunto(s)

Encéfalo/citología , Células Ependimogliales/citología , Técnicas de Ablación/métodos , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Giro Dentado/citología , Giro Dentado/metabolismo , Células Ependimogliales/metabolismo , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteína Ácida Fibrilar de la Glía/genética , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Pez Cebra

RESUMEN

BACKGROUND: Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Several reports have demonstrated that VPA functions as a histone deacetylase (HDAC) inhibitor. While VPA is known to cause teratogenic changes in the embryonic zebrafish brain, its effects on neural stem cells (NSCs) in both the embryonic and adult zebrafish are not well understood. RESULTS: In this study, we observed a proliferative effect of VPA on NSCs in the embryonic hindbrain. In contrast, VPA reduced cell proliferation in the adult zebrafish optic tectum. Treatment with HDAC inhibitors showed a similar inhibitory effect on cell proliferation in the adult zebrafish optic tectum, suggesting that VPA reduces cell proliferation through HDAC inhibition. Cell cycle progression was also suppressed in the optic tectum of the adult zebrafish brain because of HDAC inhibition. Recent studies have demonstrated that HDAC inhibits the Notch signaling pathway; hence, adult zebrafish were treated with a Notch inhibitor. This increased the number of proliferating cells in the adult zebrafish optic tectum with down-regulated expression of her4, a target of Notch signaling. CONCLUSIONS: These results suggest that VPA inhibits HDAC activity and upregulates Notch signaling to reduce cell proliferation in the optic tectum of adult zebrafish.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Células-Madre Neurales/efectos de los fármacos , Colículos Superiores/citología , Ácido Valproico/farmacología , Pez Cebra/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Western Blotting , Bromodesoxiuridina , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Cartilla de ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Etiquetado Corte-Fin in Situ , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Colículos Superiores/efectos de los fármacos , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo

RESUMEN

Dpysls (CRMPs) that were initially identified as mediator proteins of Semaphorin3a (Sema3a) signaling are involved in neuronal polarity and axon elongation in cultured neurons. Previous studies have shown that knockdown of neuropilin1a, one of the sema3a receptors, exhibited ectopic primary motor neurons (PMNs) outside of the spinal cord in zebrafish. However, downstream molecules of sema3a signaling involved in the positioning of motor neurons are largely unknown. Here, we addressed the role of Dpysl2 (CRMP2) and Dpysl3 (CRMP4) in the positioning of PMNs in the zebrafish spinal cord. We found that the knockdown of dpysls by antisense morpholino oligonucleotides (AMO) causes abnormal positioning of caudal primary (CaP) motor neurons outside the spinal cord. The knockdown of cdk5 and dyrk2 by AMO also caused similar phenotype in the positioning of CaP motor neurons, and this phenotype was rescued by co-injection of phosphorylation-mimic type dpysl2 mRNA. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and Dyrk2 is required for correct positioning of CaP motor neurons in the zebrafish spinal cord.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica/genética , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Médula Espinal/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Proteínas del Tejido Nervioso/genética , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Semaforina-3A/genética , Semaforina-3A/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Pez Cebra/genética , Proteínas de Pez Cebra/genética

RESUMEN

BACKGROUND: Sphingolipids represent a major class of lipids which both serve as structural components of membranes and as bioactive molecules involved in lipid signaling. Ceramide synthases (cers) reside in the center of sphingolipid metabolism by producing ceramide through de novo synthesis or degradative pathways. While the six mammalian cers family members have been extensively studied in cell culture and in adult tissues, a systematic analysis of cers expression and function during embryogenesis is still lacking. RESULTS: Using bioinformatic and phylogenetic analysis, we identified nine highly conserved homologs of the vertebrate cers gene family in the zebrafish genome. A systematic expression analysis throughout five developmental stages indicates that, whereas until 48 hours post fertilization most zebrafish cers homologs are expressed in distinct patterns, e.g., in the intermediate cell mass and the pronephric duct, they show a highly overlapping expression during later stages of embryonic development, mostprominently in the developing brain. CONCLUSIONS: In this study, the expression of the cers gene homologs is comprehensively analyzed for the first time during vertebrate embryogenesis. Our data indicate that each embryonic tissue has a unique profile of cers expression during zebrafish embryogenesis suggesting tissue-specific profiles of ceramides and their derivatives.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Familia de Multigenes/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Encéfalo/metabolismo , Biología Computacional , Regulación del Desarrollo de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Hibridación in Situ , Funciones de Verosimilitud , Modelos Genéticos , Especificidad de Órganos/genética , Filogenia , Pez Cebra/metabolismo

RESUMEN

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.

Asunto(s)

Quinasa 5 Dependiente de la Ciclina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/metabolismo , Neurulación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Cresta Neural/citología , Neuronas/metabolismo , Fosforilación , Pez Cebra/metabolismo , Quinasas DyrK

RESUMEN

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.

Asunto(s)

Polaridad Celular/fisiología , Proteínas del Ojo/metabolismo , Mitosis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Células Neuroepiteliales , Receptores Notch/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Movimiento Celular/fisiología , Desarrollo Embrionario/fisiología , Proteínas del Ojo/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Mutación , Proteínas del Tejido Nervioso/genética , Células Neuroepiteliales/citología , Células Neuroepiteliales/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Notch/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Madre/citología , Células Madre/fisiología , Pez Cebra/anatomía & histología , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética

RESUMEN

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.

Asunto(s)

Axones/fisiología , Proteínas de Homeodominio/fisiología , Proteínas del Tejido Nervioso/fisiología , Células Receptoras Sensoriales/fisiología , Proteínas de Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Axones/metabolismo , Secuencia de Bases , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Sitios Genéticos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM , Modelos Biológicos , Datos de Secuencia Molecular , Neuronas Motoras/metabolismo , Neuronas Motoras/fisiología , Mutación/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Receptoras Sensoriales/metabolismo , Homología de Secuencia , Nervios Espinales/anomalías , Nervios Espinales/embriología , Nervios Espinales/metabolismo , Factores de Transcripción , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo

RESUMEN

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.

Asunto(s)

Encéfalo/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Células Madre/metabolismo , Colículos Superiores/metabolismo , Adulto , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Bromodesoxiuridina/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Madre Multipotentes/metabolismo , Neuroglía/citología , Neuronas/citología , Células Madre/citología , Células Madre/fisiología , Pez Cebra/genética , Pez Cebra/metabolismo

RESUMEN

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 (twd(rw685)) mutant embryos, the vagus motor neuron progenitors stray medially away from the normal migratory pathway and fail to stop in the right location. The twd(rw685) 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 twd(rw685) 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.

Asunto(s)

Neuronas Motoras/fisiología , Células Neuroepiteliales/fisiología , Polisacáridos/fisiología , Rombencéfalo/embriología , Células Madre/fisiología , Nervio Vago/fisiología , Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo , Movimiento Celular , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Hidroliasas/genética , Datos de Secuencia Molecular , Mutación , Rombencéfalo/citología , Nervio Vago/citología , Nervio Vago/embriología , Pez Cebra/embriología

RESUMEN

In mammals, the blockade of the phototransduction cascade causes loss of vision and, in some cases, degeneration of photoreceptors. However, the molecular mechanisms that link phototransduction with photoreceptor degeneration remain to be elucidated. Here, we report that a mutation in the gene encoding a central effector of the phototransduction cascade, cGMP phosphodiesterase 6alpha'-subunit (PDE6alpha'), affects not only the vision but also the survival of cone photoreceptors in zebrafish. We isolated a zebrafish mutant, called eclipse (els), which shows no visual behavior such as optokinetic response (OKR). The cloning of the els mutant gene revealed that a missense mutation occurred in the pde6alpha' gene, resulting in a change in a conserved amino acid. The PDE6 expressed in rod photoreceptors is a heterotetramer comprising two closely related similar hydrolytic alpha and beta subunits and two identical inhibitory gamma subunits, while the PDE6 expressed in cone photoreceptors consists of two homodimers of alpha' subunits, each with gamma subunits. The els mutant displays no visual response to bright light, where cones are active, but shows relatively normal OKR to dim light, where only rods function, suggesting that only the cone-specific phototransduction pathway is disrupted in the els mutant. Furthermore, in the els mutant, cones are selectively eliminated but rods are retained at the adult stage, suggesting that cones undergo a progressive degeneration in the els mutant retinas. Taken together, these data suggest that PDE6alpha' activity is important for the survival of cones in zebrafish.

Asunto(s)

Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Regulación de la Expresión Génica , Mutación Missense , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/genética , Alelos , Secuencia de Aminoácidos , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/fisiología , Humanos , Fototransducción , Datos de Secuencia Molecular , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Bastones/patología , Células Fotorreceptoras Retinianas Bastones/fisiología , Homología de Secuencia de Aminoácido , Visión Ocular , Pez Cebra

RESUMEN

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.

Asunto(s)

Axones/fisiología , Desarrollo Embrionario/genética , Nervio Facial/embriología , Receptores de Superficie Celular/fisiología , Nervio Trigémino/embriología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Neuronas Motoras/fisiología , Mutación , Factores de Crecimiento Nervioso , Receptores de Superficie Celular/genética , Semaforinas/fisiología , Pez Cebra/genética , Proteínas de Pez Cebra/genética

RESUMEN

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.

Asunto(s)

Cadherinas/genética , Receptores Frizzled/genética , Rombencéfalo/embriología , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Cadherinas/metabolismo , Movimiento Celular/genética , Movimiento Celular/fisiología , Receptores Frizzled/metabolismo , Regulación del Desarrollo de la Expresión Génica , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Mutación , Células Neuroepiteliales/citología , Células Neuroepiteliales/metabolismo , Rombencéfalo/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo

RESUMEN

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.

Asunto(s)

Movimiento Celular/fisiología , Nervio Facial/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuronas Motoras/citología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Secuencia de Bases , Movimiento Celular/genética , Mapeo Cromosómico , Cruzamientos Genéticos , Cartilla de ADN , Gástrula/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Mutación/genética , Oligonucleótidos Antisentido , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rombencéfalo/embriología , Análisis de Secuencia de ADN

RESUMEN

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.

Asunto(s)

Tipificación del Cuerpo/fisiología , Sistema Nervioso Central/embriología , Proteínas de la Matriz Extracelular/metabolismo , Fenotipo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Proteínas Morfogenéticas Óseas/metabolismo , Mapeo Cromosómico , Cartilla de ADN , Epistasis Genética , Proteínas de la Matriz Extracelular/genética , Componentes del Gen , Genotipo , Proteínas Hedgehog , Hibridación in Situ , Datos de Secuencia Molecular , Mutación/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Pez Cebra/embriología , Proteínas de Pez Cebra/genética

RESUMEN

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.

Asunto(s)

Cadherinas/metabolismo , Prosencéfalo/embriología , Retina/embriología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Uniones Adherentes/metabolismo , Alelos , Animales , Secuencia de Bases , Tipificación del Cuerpo , Cadherinas/genética , Adhesión Celular , División Celular , ADN Complementario/genética , Mutación , Neuronas/citología , Prosencéfalo/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Retina/citología , Retina/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Transducción de Señal , Pez Cebra/genética , Proteínas de Pez Cebra/genética