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Neural crest cells generate numerous derivatives, including pigment cells, and are a model for studying how fate specification from multipotent progenitors is controlled. In mammals, the core gene regulatory network for melanocytes (their only pigment cell type) contains three transcription factors, Sox10, Pax3 and Mitf, with the latter considered a master regulator of melanocyte development. In teleosts, which have three to four pigment cell types (melanophores, iridophores and xanthophores, plus leucophores e.g. in medaka), gene regulatory networks governing fate specification are poorly understood, although Mitf function is considered conserved. Here, we show that the regulatory relationships between Sox10, Pax3 and Mitf are conserved in zebrafish, but the role for Mitf is more complex than previously emphasized, affecting xanthophore development too. Similarly, medaka Mitf is necessary for melanophore, xanthophore and leucophore formation. Furthermore, expression patterns and mutant phenotypes of pax3 and pax7 suggest that Pax3 and Pax7 act sequentially, activating mitf expression. Pax7 modulates Mitf function, driving co-expressing cells to differentiate as xanthophores and leucophores rather than melanophores. We propose that pigment cell fate specification should be considered to result from the combinatorial activity of Mitf with other transcription factors. [ABSTRACT FROM AUTHOR]

Spatio-temporal information about head orientation and movement is fundamental to the sense of balance and motion. Hair cells (HCs) in otolith organs of the vestibular system transduce linear acceleration, including head tilt and vibration. Here, we build a tiltable objective microscope in which an objective lens and specimen tilt together. With in vivo Ca2+ imaging of all utricular HCs and ganglion neurons during 360° static tilt and vibration in pitch and roll axes, we reveal the direction- and static/dynamic stimulus-selective topographic responses in larval zebrafish. We find that head vibration is preferentially received by striolar HCs, whereas static tilt is preferentially transduced by extrastriolar HCs. Spatially ordered direction preference in HCs is consistent with hair-bundle polarity and is preserved in ganglion neurons through topographic innervation. Together, these results demonstrate topographically organized selectivity for direction and dynamics of head orientation/movement in the vestibular periphery. Signals about head orientation and movement in the vestibular periphery are fundamental to the sense of balance and motion, but difficult to measure systematically during head motion. Here, the authors build a microscope that visualizes neural activity in hair cells and vestibular ganglion cells during 360° head tilt and vibration in zebrafish larvae, and reveal a topographic organization of direction- and static/dynamic stimulus-selective responses. [ABSTRACT FROM AUTHOR]

Developmental maturation occurs in slow swimming behavior in larval zebrafish; older larvae acquire the ability to perform slow swimming while keeping their head stable in the yaw dimension. A class of long-distance descending commissural excitatory V0v neurons, called MCoD neurons, are known to develop in a later phase of neurogenesis, and participate in slow swimming in older larvae. We hypothesized that these MCoD neurons play a role in coordinating the activities of trunk muscles in the diagonal dimension (e.g., the rostral left and the caudal right) to produce the S-shaped swimming form that contributes to the stability of the head. Here, we show that MCoD neurons do indeed play this role. In larvae in which MCoD neurons were laser-ablated, the swimming body form often adopted a one-sided (C-shaped) bend with reduced appearance of the normal S-shaped bend. With this change in swimming form, the MCoD-ablated larvae exhibited a greater degree of head yaw displacement during slow swimming. In mice, the long-distance descending commissural V0v neurons have been implicated in diagonal interlimb coordination during walking. Together with this, our study suggests that the long-distance descending commissural V0v neurons form an evolutionarily conserved pathway in the spinal locomotor circuits that coordinates the movements of the diagonal body/limb muscles. [ABSTRACT FROM AUTHOR]

The most basic form of locomotion in limbed vertebrates consists of alternating activities of the flexor and extensor muscles within each limb coupled with left/right limb alternation. Although larval zebrafish are not limbed, their pectoral fin movements exhibit the following fundamental aspects of this basic movement: abductor/adductor alternation (corresponding to flexor/extensor alternation) and left/right fin alternation. Because of the simplicity of their movements and the compact neural organization of their spinal cords, zebrafish can serve as a good model to identify the neuronal networks of the central pattern generator (CPG) that controls rhythmic appendage movements. Here, we set out to investigate neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish, using transgenic fish that specifically express GFP in abductor or adductor motor neurons (MNs) and candidate CPG neurons. First, we showed that spiking activities of abductor and adductor MNs were essentially alternating. Second, both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively, indicating that the MN spiking activities are controlled in a push-pull manner. Further, we obtained the following evidence that dmrt3a-expressing commissural inhibitory neurons are involved in regulating the activities of abductor MNs: (1) strong inhibitory synaptic connections were found from dmrt3a neurons to abductor MNs; and (2) ablation of dmrt3a neurons shifted the spike timing of abductor MNs. Thus, in this simple system of abductor/adductor alternation, the last-order inhibitory inputs originating from the contralaterally located neurons play an important role in controlling the firing timings of MNs. [ABSTRACT FROM AUTHOR]

During many behaviors in vertebrates, the CNS generates asymmetric activities between the left and right sides to produce asymmetric body movements. For asymmetrical activations of the CNS, reciprocal inhibition between the left and right sides is believed to play a key role. However, the complexity of the CNS makes it difficult to identify the reciprocal inhibition circuits at the level of individual cells and the contribution of each neuron to the asymmetric activity. Using larval zebrafish, we examined this issue by investigating reciprocal inhibition circuits between a pair of Mauthner (M) cells, giant reticulospinal neurons that trigger fast escapes. Previous studies have shown that a class of excitatory neurons, called cranial relay neurons, is involved in the reciprocal inhibition pathway between the M cells. Using transgenic fish, in which two of the cranial relay neurons (Ta1 and Ta2) expressed GFP, we showed that Ta1 and Ta2 constitute major parts of the pathway. In larvae in which Ta1/Ta2 were laser-ablated, the amplitude of the reciprocal IPSPs dropped to less than one-third. Calcium imaging and electrophysiological recording showed that the occurrence probability of bilateral M-cell activation upon sound/vibration stimuli was greatly increased in the Ta1/Ta2-ablated larvae. Behavioral experiments revealed that the Ta1/Ta2 ablation resulted in shallower body bends during sound/vibration-evoked escapes, which is consistent with the observation that increased occurrence of bilateral M-cell activation impaired escape performance. Our study revealed major components of the reciprocal inhibition circuits in the M cell system and the behavioral importance of the circuits. [ABSTRACT FROM AUTHOR]

Background: The neurons in the vertebrate retina arise from multipotent retinal progenitor cells (RPCs). It is not clear, however, which progenitors are multipotent or why they are multipotent. Results: In this study we show that the homeodomain transcription factor Vsx2 is initially expressed throughout the retinal epithelium, but later it is downregulated in all but a minor population of bipolar cells and all Müller glia. The Vsx2-negative daughters of Vsx2-positive RPCs divide and give rise to all other cell types in the retina. Vsx2 is a repressor whose targets include transcription factors such as Vsx1, which is expressed in the progenitors of distinct non-Vsx2 bipolars, and the basic helix-loop-helix transcription factor Ath5, which restricts the fate of progenitors to retinal ganglion cells, horizontal cells, amacrine cells and photoreceptors fates. Foxn4, expressed in the progenitors of amacrine and horizontal cells, is also negatively regulated by Vsx2. Conclusion: Our data thus suggest Vsx2-positive RPCs are fully multipotent retinal progenitors and that when Vsx2 is downregulated, Vsx2-negative progenitors escape Vsx2 repression and so are able to express factors that restrict lineage potential. [ABSTRACT FROM AUTHOR]

The zebrafish dorsal habenula (dHb) shows conspicuous asymmetry in its connection with the interpeduncular nucleus (IPN) and is equivalent to the mammalian medial habenula. Genetic inactivation of the lateral subnucleus of dHb (dHbL) biased fish towards freezing rather than the normal flight response to a conditioned fear stimulus, suggesting that the dHbL-IPN pathway is important for controlling experience-dependent modification of fear responses. [ABSTRACT FROM AUTHOR]

The zebrafish is an important model in systems neuroscience but viral tools to dissect the structure and function of neuronal circuitry are not established. We developed methods for efficient gene transfer and retrograde tracing in adult and larval zebrafish by herpes simplex viruses (HSV1). HSV1 was combined with the Gal4/UAS system to target cell types with high spatial, temporal, and molecular specificity. We also established methods for efficient transneuronal tracing by modified rabies viruses in zebrafish. We demonstrate that HSV1 and rabies viruses can be used to visualize and manipulate genetically or anatomically identified neurons within and across different brain areas of adult and larval zebrafish. An expandable library of viruses is provided to express fluorescent proteins, calcium indicators, optogenetic probes, toxins and other molecular tools. This toolbox creates new opportunities to interrogate neuronal circuits in zebrafish through combinations of genetic and viral approaches. [ABSTRACT FROM AUTHOR]

As sensory information moves through the brain, higher-order areas exhibit more complex tuning than lower areas. Though models predict that complexity arises via convergent inputs from neurons with diverse response properties, in most vertebrate systems, convergence has only been inferred rather than tested directly. Here, we measure sensory computations in zebrafish vestibular neurons across multiple axes in vivo. We establish that whole-cell physiological recordings reveal tuning of individual vestibular afferent inputs and their postsynaptic targets. Strong, sparse synaptic inputs can be distinguished by their amplitudes, permitting analysis of afferent convergence in vivo. An independent approach, serial-section electron microscopy, supports the inferred connectivity. We find that afferents with similar or differing preferred directions converge on central vestibular neurons, conferring more simple or complex tuning, respectively. Together, these results provide a direct, quantifiable demonstration of feedforward input convergence in vivo. • Recorded tuning of both vestibular inputs and their central targets in zebrafish • Similarly tuned inputs tended to converge together • Less frequently, inputs with different tuning converged postsynaptically • Postsynaptic tuning could largely be explained as a summation of excitatory inputs Though computational models predict how sensory inputs converge in central neurons, it is technically challenging to test these ideas experimentally. With whole-cell recordings in larval zebrafish in vivo , Liu et al. demonstrate that the convergence of similarly or differently tuned vestibular afferents produces more simple or complex postsynaptic tuning, respectively. [ABSTRACT FROM AUTHOR]

Motor systems must continuously adapt their output to maintain a desired trajectory. While the spinal circuits underlying rhythmic locomotion are well described, little is known about how the network modulates its output strength. A major challenge has been the difficulty of recording from spinal neurons during behavior. Here, we use voltage imaging to map the membrane potential of large populations of glutamatergic neurons throughout the spinal cord of the larval zebrafish during fictive swimming in a virtual environment. We characterized a previously undescribed subpopulation of tonic-spiking ventral V3 neurons whose spike rate correlated with swimming strength and bout length. Optogenetic activation of V3 neurons led to stronger swimming and longer bouts but did not affect tail beat frequency. Genetic ablation of V3 neurons led to reduced locomotor adaptation. The power of voltage imaging allowed us to identify V3 neurons as a critical driver of locomotor adaptation in zebrafish. • Voltage imaging reveals the dynamics of zebrafish spinal motor circuits • A ventral glutamatergic neuron population modulates swim strength • Tail beat strength and bout duration are controlled separately from beat frequency Böhm and co-workers apply light-sheet voltage imaging to the spinal cords of zebrafish swimming in a virtual environment and identify a subpopulation of glutamatergic neurons that modulates swim strength and bout duration without affecting tail beat frequency. [ABSTRACT FROM AUTHOR]