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Expression of halorhodopsin (NpHR), a light-driven microbial chloride pump, enables optical control of membrane potential and reversible silencing of targeted neurons. We generated transgenic zebrafish expressing enhanced NpHR under control of the Gal4/UAS system. Electrophysiological recordings showed that eNpHR stimulation effectively suppressed spiking of single neurons in vivo. Applying light through thin optic fibers positioned above the head of a semi-restrained zebrafish larva enabled us to target groups of neurons and to simultaneously test the effect of their silencing on behavior. The photostimulated volume of the zebrafish brain could be marked by subsequent photoconversion of co-expressed Kaede or Dendra. These techniques were used to localize swim command circuitry to a small hindbrain region, just rostral to the commissura infima Halleri. The kinetics of the hindbrain-generated swim command was investigated by combined and separate photo-activation of NpHR and Channelrhodopsin-2 (ChR2), a light-gated cation channel, in the same neurons. Together this \"optogenetic toolkit\" allows loss-of-function and gain-of-function analyses of neural circuitry at high spatial and temporal resolution in a behaving vertebrate.

Large, living biological specimens present challenges to existing optical imaging techniques because of their absorptive and scattering properties. We developed selective plane illumination microscopy (SPIM) to generate multidimensional images of samples up to a few millimeters in size. The system combines two-dimensional illumination with orthogonal camera-based detection to achieve high-resolution, optically sectioned imaging throughout the sample, with minimal photodamage and at speeds capable of capturing transient biological phenomena. We used SPIM to visualize all muscles in vivo in the transgenic Medaka line Arnie, which expresses green fluorescent protein in muscle tissue. We also demonstrate that SPIM can be applied to visualize the embryogenesis of the relatively opaque Drosophila melanogaster in vivo.

The optic tectum of zebrafish is involved in behavioral responses that require the detection of small objects. The superficial layers of the tectal neuropil receive input from retinal axons, while its deeper layers convey the processed information to premotor areas. Imaging with a genetically encoded calcium indicator revealed that the deep layers, as well as the dendrites of single tectal neurons, are preferentially activated by small visual stimuli. This spatial filtering relies on GABAergic interneurons (using the neurotransmitter γ-aminobutyric acid) that are located in the superficial input layer and respond only to large visual stimuli. Photo-ablation of these cells with KillerRed, or silencing of their synaptic transmission, eliminates the size tuning of deeper layers and impairs the capture of prey.

While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA protospacer adjacent motif, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic an mutation of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes including cbl . With this last target, we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease-associated mutations.

While the exterior of vertebrate bodies appears bilaterally symmetrical, internal organ positioning and morphology frequently exhibit left-right (L-R) asymmetries. In several vertebrates, including human, mouse, frog, and zebrafish, left-right symmetry-breaking during embryonic development is initiated by a ciliated organ called the Node or left-right organizer. Within the Node, a leftward flow of extraembryonic fluid named the Nodal flow mediates the asymmetric expressions of Nodal factors. Although downstream Nodal pathway components leading to the establishment of the embryonic left-right axis are well known, less is known about the development and formation of the embryonic Node itself. Here, we reveal a novel role for the Meteorin protein family in the establishment of the left-right axis and in the formation of Kupffer’s vesicle, the Node equivalent structure in zebrafish. We show that the genetic inactivation of each or all three members of the zebrafish Meteorin family ( metrn , metrn-like a, and metrn-like b ) leads to defects in properties of the Kupffer’s vesicle, caused by impaired assembly and migration of the Kupffer’s vesicle forming dorsal forerunner cells. In addition, we demonstrate that Meteorins genetically interact with integrins ItgαV and Itgβ1b, regulating the dorsal forerunner cell clustering, and that meteorins loss-of-function results in disturbed Nodal factor expression and consequently in randomized or symmetric heart looping and jogging. These results identify a new role for the Meteorin protein family in the left-right asymmetry patterning during embryonic vertebrate development.

Axon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. We have identified N-myc downstream-regulated gene 4, ndrg4, as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 and nsf are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery.

Organogenesis in vertebrates requires the tight control of cell proliferation and differentiation. The homeobox-containing transcription factor Six3 plays a pivotal role 1 , 2 in the proliferation of retinal precursor cells. In a yeast two-hybrid screen, we identified the DNA replication-inhibitor geminin as a partner of Six3. Geminin inhibits cell-cycle progression 3 by sequestering Cdt1 (refs 4 , 5 ), the key component for the assembly of the pre-replication complex 6 . Here, we show that Six3 efficiently competes with Cdt1 directly to bind to geminin, which reveals how Six3 can promote cell proliferation without transcription. In common with Six3 inactivation 2 , 7 , overexpression of the geminin gene ( Gem ; also known as Gmn ) in medaka ( Oryzias latipes ) induces specific forebrain and eye defects that are rescued by Six3 . Conversely, loss of Gem (in common with gain of Six3 (ref. 1 )) promotes retinal precursor-cell proliferation and results in expanded optic vesicles, markedly potentiating Six3 gain-of-function phenotypes. Our data indicate that the transcription factor Six3 and the replication-initiation inhibitor geminin act antagonistically to control the balance between proliferation and differentiation during early vertebrate eye development.

Reelin (Reln) is an extracellular glycoprotein that is important for brain patterning. During development Reln coordinates the radial migration of postmitotic cortical neurons, cerebellar and hippocampal neurons, whereas it promotes dendrite maturation, synaptogenesis, synaptic transmission, plasticity and neurotransmitter release in the postnatal and adult brain. Genetic studies of human patients have demonstrated association between the RELN locus and autism spectrum disorder, schizophrenia, bipolar disorder, and Alzheimer's disease. In this study we have characterized the behavioral phenotype of ( ) mutant zebrafish, as well as two canonical signaling pathway targets ( ) and the ( ). Zebrafish mutants display a selective reduction in preference for social novelty that is not observed in or mutant lines. They also exhibit an increase in 5-HT signaling in the hindbrain that parallels but does not underpin the alteration in social preference. These results suggest that zebrafish mutants can be used to model some aspects of human diseases in which changes to Reln signaling alter social behavior.

Development and function of highly polarized cells such as neurons depend on microtubule-associated intracellular transport, but little is known about contributions of specific molecular motors to the establishment of synaptic connections. In this study, we investigated the function of the Kinesin I heavy chain Kif5aa during retinotectal circuit formation in zebrafish. Targeted disruption of Kif5aa does not affect retinal ganglion cell differentiation, and retinal axons reach their topographically correct targets in the tectum, albeit with a delay. In vivo dynamic imaging showed that anterograde transport of mitochondria is impaired, as is synaptic transmission. Strikingly, disruption of presynaptic activity elicits upregulation of Neurotrophin-3 (Ntf3) in postsynaptic tectal cells. This in turn promotes exuberant branching of retinal axons by signaling through the TrkC receptor (Ntrk3). Thus, our study has uncovered an activity-dependent, retrograde signaling pathway that homeostatically controls axonal branching. Different regions of a neuron have distinct structures and roles. For example, each neuron has a cable-like structure called the axon that extends out of the body of the cell and carries electrical signals away from the cell body. To pass these messages on to neighboring cells, branches on the axon form connections called synapses with other neurons. The axon lacks most of the cellular machinery needed to make proteins and other molecules that the cell needs to work correctly. Therefore, neurons must transport these materials from the cell body—where they are produced—down to the end of the axon. Specialized proteins called molecular motors carry this cargo down the axon along ‘tracks’ composed of filaments called microtubules. Auer, Xiao et al. have now used genetic techniques to disrupt the gene that encodes an important molecular motor, called Kif5A, in developing zebrafish larvae. The effects of this manipulation on the development of the zebrafish's visual system were then examined. When zebrafish are a few days old, neurons in the retina—the structure at the back of the eye that responds to light—extend axons into a region of the brain called the tectum. The formation of synapses between cells in the retina and the tectum provides a pathway that enables information to travel from the eye to the brain. Auer, Xiao et al. found that in larvae that lack Kif5A, axons from the retina enter the brain about a day later than they do in normal larvae. However, when these mutant axons arrive, they produce large numbers of branches, each with the potential to form multiple synapses with cells in the tectum. However, none of the resulting synapses appear to respond to visual stimuli, which is consistent with the fact that Kif5A mutant larvae are blind. Experiments to identify what triggers the excessive branching of retinal axons revealed that the mutant fish had elevated levels of a growth-promoting protein called neurotrophin-3 in cells in the tectum. This increased production of neurotrophin-3 was also observed when neuronal activity was blocked, for example by toxins. The lack of neuronal activity in retinal axons therefore seems to increase the production of neurotrophin-3, which in turn stimulates axonal branching. Future experiments could investigate the molecular signal that drives this increased production of neurotrophin-3, and how this is regulated during normal neuronal development.

Kolmer–Agduhr cells in spinal cord development In the brief period during which we have known of their existence, light-gated ion channels have been used to assess the function of known cell types to which they are genetically targeted. Here Wyart et al . search for unknown cell types that drive the central pattern generator of locomotion. GAL4 lines of zebrafish in which light-gated glutamate receptors were sparsely expressed in diverse, partially overlapping sets of neurons were screened. Common behavioural effects of light could thus be attributed to activity in a specific cell type when it is the only cell shared between the different lines. The photo-stimulation of one specific cell type, the Kolmer–Agduhr cell, was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. Genetically silencing Kolmer–Agduhr cells reduced the frequency of spontaneous free swimming, indicating that Kolmer–Agduhr cell activity provides necessary tone for spontaneous forward swimming. Kolmer–Agduhr cells have been known for over 75 years, but their function has been mysterious. This work shows that during early development in low vertebrates these cells provide a positive drive to the spinal central pattern generator for spontaneous locomotion. In vertebrates, the excitatory synaptic drive for inducing spinal central pattern generators (CPGs) — which are responsible for generating rhythmic movements — can originate from either supraspinal glutamatergic inputs or from within the spinal cord. A spinal input to the CPG is now identified using a combination of intersectional gene expression and optogenetics in zebrafish larvae; the results reveal that during early development Kolmer–Agduhr cells provide a positive drive to the spinal CPG for spontaneous locomotion. Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements 1 . In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord 2 , 3 . Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics 4 in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer–Agduhr cell 5 , which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments 6 . Genetically silencing Kolmer–Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer–Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer–Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.