RESUMEN
The function of neuronal networks is determined not only by synaptic connectivity but also by neuromodulatory systems that broadcast information via distributed connections and volume transmission. To understand the molecular constraints that organize neuromodulatory signaling in the telencephalon of adult zebrafish, we used transcriptomics and additional approaches to delineate cell types, to determine their phylogenetic conservation, and to map the expression of marker genes at high granularity. The combinatorial expression of GPCRs and cell-type markers indicates that all neuronal cell types are subject to modulation by multiple monoaminergic systems and distinct combinations of neuropeptides. Individual cell types were associated with multiple (typically >30) neuromodulatory signaling networks but expressed only a few diagnostic GPCRs at high levels, suggesting that different neuromodulatory systems act in combination, albeit with unequal weights. These results provide a detailed map of cell types and brain areas in the zebrafish telencephalon, identify core components of neuromodulatory networks, highlight the cell-type specificity of neuropeptides and GPCRs, and begin to decipher the logic of combinatorial neuromodulation.
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Neuropéptidos , Pez Cebra , Animales , Pez Cebra/fisiología , Filogenia , Neuronas/metabolismo , Telencéfalo , Neuropéptidos/metabolismoRESUMEN
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.
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Virus de la Rabia , Pez Cebra , Animales , Expresión Génica , Neuronas/fisiología , Optogenética/métodos , Virus de la Rabia/genética , Pez Cebra/genéticaRESUMEN
In this issue of Neuron, Gurnani and Silver (2021) report that activity across Golgi cells, a major type of inhibitory interneuron in the cerebellar cortex, is multidimensional and modulated by behavior. These results suggest multiple functions for inhibition in cerebellar computations.
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Corteza Cerebelosa , Interneuronas , Cerebelo , NeuronasRESUMEN
Commissural inhibitory neurons in the spinal cord of aquatic vertebrates coordinate left-right body alternation during swimming. Their developmental origin, however, has been elusive. We investigate this by comparing the anatomy and function of two commissural inhibitory neuron types, dI6dmrt3a and V0d, derived from the pd6 and p0 progenitor domains, respectively. We find that both of these commissural neuron types have monosynaptic, inhibitory connections to neuronal populations active during fictive swimming, supporting their role in providing inhibition to the contralateral side. V0d neurons tend to fire during faster and stronger movements, while dI6dmrt3a neurons tend to fire more consistently during normal fictive swimming. Ablation of dI6dmrt3a neurons leads to an impairment of left-right alternating activity through abnormal co-activation of ventral root neurons on both sides of the spinal cord. Our results suggest that dI6dmrt3a and V0d commissural inhibitory neurons synergistically provide inhibition to the opposite side across different swimming behaviors.
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Glicina/metabolismo , Inhibición Neural/fisiología , Neuronas/fisiología , Pez Cebra/fisiología , Animales , Eliminación de Gen , Proteínas Fluorescentes Verdes/metabolismo , Potenciales Postsinápticos Inhibidores , Larva , Locomoción , Fenotipo , Médula Espinal/fisiología , Natación , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Intelligent behavior involves associations between high-dimensional sensory representations and behaviorally relevant qualities such as valence. Learning of associations involves plasticity of excitatory connectivity, but it remains poorly understood how information flow is reorganized in networks and how inhibition contributes to this process. We trained adult zebrafish in an appetitive odor discrimination task and analyzed odor representations in a specific compartment of the posterior zone of the dorsal telencephalon (Dp), the homolog of mammalian olfactory cortex. Associative conditioning enhanced responses with a preference for the positively conditioned odor. Moreover, conditioning systematically remapped odor representations along an axis in coding space that represented attractiveness (valence). Interindividual variations in this mapping predicted variations in behavioral odor preference. Photoinhibition of interneurons resulted in specific modifications of odor representations that mirrored effects of conditioning and reduced experience-dependent, interindividual variations in odor-valence mapping. These results reveal an individualized odor-to-valence map that is shaped by inhibition and reorganized during learning.
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Aprendizaje por Asociación/fisiología , Mapeo Encefálico , Inhibición Neural/fisiología , Corteza Olfatoria/fisiología , Percepción Olfatoria/fisiología , Animales , Animales Modificados Genéticamente , Aprendizaje Discriminativo/fisiología , Femenino , Individualidad , Masculino , Odorantes , Pez CebraRESUMEN
The developing nervous system consists of a variety of cell types. Transgenic animals expressing reporter genes in specific classes of neuronal cells are powerful tools for the study of neuronal network formation. We generated a wide variety of transgenic zebrafish that expressed reporter genes in specific classes of neurons or neuronal progenitors. These include lines in which neurons of specific neurotransmitter phenotypes expressed fluorescent proteins or Gal4, and lines in which specific subsets of the dorsal progenitor domain in the spinal cord expressed fluorescent proteins. Using these, we examined domain organization in the developing dorsal spinal cord, and found that there are six progenitor domains in zebrafish, which is similar to the domain organization in mice. We also systematically characterized neurotransmitter properties of the neurons that are produced from each domain. Given that reporter gene expressions occurs in a wide area of the nervous system in the lines generated, these transgenic fish should serve as powerful tools for the investigation of not only the neurons in the dorsal spinal cord but also neuronal structures and functions in many other regions of the nervous system.
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Técnicas de Transferencia de Gen , Neuronas/citología , Neuronas/metabolismo , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Ratones , Neurotransmisores/metabolismo , Fenotipo , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
BACKGROUND: During locomotion in vertebrates, reticulospinal neurons in the hindbrain play critical roles in providing descending excitation to the spinal cord locomotor systems. However, despite the fact that many genes that are used to classify the neuronal identities of neurons in the hindbrain have been identified, the molecular identity of the reticulospinal neurons that are critically involved in locomotor drive is not well understood. Chx10-expressing neurons (V2a neurons) are ipsilaterally projecting glutamatergic neurons in the spinal cord and the hindbrain. Many of the V2a neurons in the hindbrain are known to project to the spinal cord in zebrafish, making hindbrain V2a neurons a prime candidate in descending locomotor drive. RESULTS: We investigated the roles of hindbrain V2a neurons using optogenetic and electrophysiological approaches. The forced activation of hindbrain V2a neurons using channelrhodopsin efficiently evoked swimming, whereas the forced inactivation of them using Archearhodopsin3 or Halorhodpsin reliably stopped ongoing swimming. Electrophysiological recordings of two populations of hindbrain reticulospinal V2a neurons showed that they were active during swimming. One population of neurons, small V2a neurons in the caudal hindbrain, fired with low rhythmicity, whereas the other population of neurons, large reticulospinal V2a neurons, called MiV1 neurons, fired more rhythmically. CONCLUSIONS: These results indicated that hindbrain reticulospinal V2a neurons play critical roles in providing excitation to the spinal locomotor circuits during swimming by providing both tonic and phasic inputs to the circuits.
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Locomoción , Neuronas/fisiología , Rombencéfalo/fisiología , Médula Espinal/fisiología , Natación , Pez Cebra/fisiología , AnimalesRESUMEN
The developing spinal cord is subdivided into distinct progenitor domains, each of which gives rise to different types of neurons. However, the developmental mechanisms responsible for generating neuronal diversity within a domain are not well understood. Here, we have studied zebrafish V0 neurons, those that derive from the p0 progenitor domain, to address this question. We find that all V0 neurons have commissural axons, but they can be divided into excitatory and inhibitory classes. V0 excitatory neurons (V0-e) can be further categorized into three groups based on their axonal trajectories; V0-eA (ascending), V0-eB (bifurcating), and V0-eD (descending) neurons. By using time-lapse imaging of p0 progenitors and their progeny, we show that inhibitory and excitatory neurons are produced from different progenitors. We also demonstrate that V0-eA neurons are produced from distinct progenitors, while V0-eB and V0-eD neurons are produced from common progenitors. We then use birth-date analysis to reveal that V0-eA, V0-eB, and V0-eD neurons arise in this order. By perturbing Notch signaling and accelerating neuronal differentiation, we predictably alter the generation of early born V0-e neurons at the expense of later born ones. These results suggest that multiple types of V0 neurons are produced by two distinct mechanisms; from heterogeneous p0 progenitors and from the same p0 progenitor, but in a time-dependent manner.
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Diferenciación Celular/fisiología , Neuronas/clasificación , Neuronas/citología , Médula Espinal/embriología , Médula Espinal/crecimiento & desarrollo , Células Madre/fisiología , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Neurogénesis/fisiología , Neuronas/fisiología , Médula Espinal/citología , Factores de Tiempo , Imagen de Lapso de Tiempo/métodosRESUMEN
BACKGROUND: Because of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals), supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans. RESULTS: In an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an egfp transgenic stable fish line that trapped tnks1bp1, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(tnks1bp1:EGFP) line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells. CONCLUSIONS: We present a Tg(tnks1bp1:EGFP) stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general.
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Animales Modificados Genéticamente , Proteínas Fluorescentes Verdes/genética , Sistema de la Línea Lateral/citología , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Transcriptoma , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Expresión Génica , Genes Reporteros , Marcadores Genéticos , Proteínas Fluorescentes Verdes/biosíntesis , Hibridación in Situ , Larva/citología , Larva/metabolismo , Sistema de la Línea Lateral/crecimiento & desarrollo , Datos de Secuencia Molecular , Mutagénesis Insercional , Mucosa Olfatoria/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteína 1 de Unión a Repeticiones Teloméricas/química , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Transcripción Genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismoRESUMEN
Bilateral symmetric tissues must interpret axial references to maintain their global architecture during growth or repair. The regeneration of hair cells in the zebrafish lateral line, for example, forms a vertical midline that bisects the neuromast epithelium into perfect mirror-symmetric plane-polarized halves. Each half contains hair cells of identical planar orientation but opposite to that of the confronting half. The establishment of bilateral symmetry in this organ is poorly understood. Here, we show that hair-cell regeneration is strongly directional along an axis perpendicular to that of epithelial planar polarity. We demonstrate compartmentalized Notch signaling in neuromasts, and show that directional regeneration depends on the development of hair-cell progenitors in polar compartments that have low Notch activity. High-resolution live cell tracking reveals a novel process of planar cell inversions whereby sibling hair cells invert positions immediately after progenitor cytokinesis, demonstrating that oriented progenitor divisions are dispensable for bilateral symmetry. Notwithstanding the invariably directional regeneration, the planar polarization of the epithelium eventually propagates symmetrically because mature hair cells move away from the midline towards the periphery of the neuromast. We conclude that a strongly anisotropic regeneration process that relies on the dynamic stabilization of progenitor identity in permissive polar compartments sustains bilateral symmetry in the lateral line.
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Tipificación del Cuerpo/genética , Compartimento Celular/fisiología , Células Epiteliales/fisiología , Receptores Notch/metabolismo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/fisiología , Polaridad Celular/genética , Polaridad Celular/fisiología , Rastreo Celular/métodos , Embrión no Mamífero , Células Epiteliales/metabolismo , Células Ciliadas Auditivas/fisiología , Sistema de la Línea Lateral/embriología , Sistema de la Línea Lateral/metabolismo , Modelos Biológicos , Receptores Notch/genética , Receptores Notch/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Imagen de Lapso de Tiempo/métodos , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
The hindbrain of larval zebrafish contains a relatively simple ground plan in which the neurons throughout it are arranged into stripes that represent broad neuronal classes that differ in transmitter identity, morphology, and transcription factor expression. Within the stripes, neurons are stacked continuously according to age as well as structural and functional properties, such as axonal extent, input resistance, and the speed at which they are recruited during movements. Here we address the question of how particular networks among the many different sensory-motor networks in hindbrain arise from such an orderly plan. We use a combination of transgenic lines and pairwise patch recording to identify excitatory and inhibitory interneurons in the hindbrain network for escape behaviors initiated by the Mauthner cell. We map this network onto the ground plan to show that an individual hindbrain network is built by drawing components in predictable ways from the underlying broad patterning of cell types stacked within stripes according to their age and structural and functional properties. Many different specialized hindbrain networks may arise similarly from a simple early patterning.
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Tipificación del Cuerpo/fisiología , Mapeo Encefálico/métodos , Reacción de Fuga/fisiología , Red Nerviosa , Rombencéfalo/fisiología , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Electrofisiología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Interneuronas/metabolismo , Larva/anatomía & histología , Larva/fisiología , Técnicas de Placa-Clamp , Desempeño Psicomotor/fisiología , Rombencéfalo/anatomía & histología , Pez Cebra/anatomía & histologíaRESUMEN
The vertebrate hindbrain contains various sensory-motor networks controlling movements of the eyes, jaw, head, and body. Here we show that stripes of neurons with shared neurotransmitter phenotype that extend throughout the hindbrain of young zebrafish reflect a broad underlying structural and functional patterning. The neurotransmitter stripes contain cell types with shared gross morphologies and transcription factor markers. Neurons within a stripe are stacked systematically by extent and location of axonal projections, input resistance, and age, and are recruited along the axis of the stripe during behavior. The implication of this pattern is that the many networks in hindbrain are constructed from a series of neuronal components organized into stripes that are ordered from top to bottom according to a neuron's age, structural and functional properties, and behavioral roles. This simple organization probably forms a foundation for the construction of the networks underlying the many behaviors produced by the hindbrain.
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Tipificación del Cuerpo/fisiología , Interneuronas/metabolismo , Modelos Neurológicos , Red Nerviosa , Neurotransmisores/metabolismo , Rombencéfalo/anatomía & histología , Pez Cebra/anatomía & histología , Factores de Edad , Animales , Animales Modificados Genéticamente , Calcio/metabolismo , Electrofisiología , Interneuronas/citología , Microscopía Confocal , Rombencéfalo/fisiología , Pez Cebra/fisiologíaRESUMEN
In mammals, cerebellar neurons are categorized as glutamatergic or GABAergic, and are derived from progenitors that express the proneural genes atoh1 or ptf1a, respectively. In zebrafish, three atoh1 genes, atoh1a, atoh1b, and atoh1c, are expressed in overlapping but distinct expression domains in the upper rhombic lip (URL): ptf1a is expressed exclusively in the ventricular zone (VZ). Using transgenic lines expressing fluorescent proteins under the control of the regulatory elements of atoh1a and ptf1a, we traced the lineages of the cerebellar neurons. The atoh1(+) progenitors gave rise not only to granule cells but also to neurons of the anteroventral rhombencephalon. The ptf1a(+) progenitors generated Purkinje cells. The olig2(+) eurydendroid cells, which are glutamatergic, were derived mostly from ptf1a(+) progenitors in the VZ but some originated from the atoh1(+) progenitors in the URL. In the adult cerebellum, atoh1a, atoh1b, and atoh1c are expressed in the molecular layer of the valvula cerebelli and of the medial corpus cerebelli, and ptf1a was detected in the VZ. The proneural gene expression patterns coincided with the sites of proliferating neuronal progenitors in the adult cerebellum. Our data indicate that proneural gene-linked neurogenesis is evolutionarily conserved in the cerebellum among vertebrates, and that the continuously generated neurons help remodel neural circuits in the adult zebrafish cerebellum.
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Cerebelo/embriología , Neurogénesis/genética , Neuronas/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Proteínas de Pez Cebra/metabolismoRESUMEN
The lateral line system displays highly divergent patterns in adult teleost fish. The mechanisms underlying this variability are poorly understood. Here, we demonstrate that the lateral line mechanoreceptor, the neuromast, gives rise to a series of accessory neuromasts by a serial budding process during postembryonic development in zebrafish. We also show that accessory neuromast formation is highly correlated to the development of underlying dermal structures such as bones and scales. Abnormalities in opercular bone morphogenesis, in endothelin 1-knockdown embryos, are accompanied by stereotypic errors in neuromast budding and positioning, further demonstrating the tight correlation between the patterning of neuromasts and of the underlying dermal bones. In medaka, where scales form between peridermis and opercular bones, the lateral line displays a scale-specific pattern which is never observed in zebrafish. These results strongly suggest a control of postembryonic neuromast patterns by underlying dermal structures. This dermal control may explain some aspects of the evolution of lateral line patterns.
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Tipificación del Cuerpo , Sistema de la Línea Lateral/crecimiento & desarrollo , Morfogénesis , Oryzias/crecimiento & desarrollo , Pez Cebra/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Huesos/embriología , Dermis/citología , Dermis/crecimiento & desarrollo , Embrión no Mamífero , Inmunohistoquímica , Hibridación in Situ , Sistema de la Línea Lateral/citología , Mecanorreceptores/citología , Microinyecciones , Microscopía por Video , Modelos Biológicos , Oligonucleótidos Antisentido/metabolismo , Oryzias/embriología , Especificidad de la Especie , Pez Cebra/embriologíaRESUMEN
In teleost fish, the Mauthner (M) cell, a large reticulospinal neuron in the brainstem, triggers escape behavior. Spinal commissural inhibitory interneurons that are electrotonically excited by the M-axon have been identified, but the behavioral roles of these neurons have not yet been addressed. Here, we studied these neurons, named CoLo (commissural local), in larval zebrafish using an enhancer-trap line in which the entire population of CoLos was visualized by green fluorescent protein. CoLos were present at one cell per hemi-segment. Electrophysiological recordings showed that an M-spike evoked a spike in CoLos via electrotonic transmission and that CoLos made monosynaptic inhibitory connections onto contralateral primary motoneurons, consistent with the results in adult goldfish. We further showed that CoLos were active only during escapes. We examined the behavioral roles of CoLos by investigating escape behaviors in CoLo-ablated larvae. The results showed that the escape behaviors evoked by sound/vibration stimuli were often impaired with a reduced initial bend of the body, indicating that CoLos play important roles in initiating escapes. We obtained several lines of evidence that strongly suggested that the impaired escapes occurred during bilateral activation of the M-cells: in normal larvae, CoLo-mediated inhibitory circuits enable animals to perform escapes even in these occasions by silencing the output of the slightly delayed firing of the second M-cell. This study illustrates (1) a clear example of the behavioral role of a specialized class of interneurons and (2) the capacity of the spinal circuits to filter descending commands and thereby produce the appropriate behavior.
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Reacción de Fuga/fisiología , Interneuronas/fisiología , Inhibición Neural/fisiología , Médula Espinal/citología , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Conducta Animal , Biotina/análogos & derivados , Biotina/metabolismo , Calcio/metabolismo , Dextranos/metabolismo , Estimulación Eléctrica , Uniones Comunicantes/fisiología , Proteínas Fluorescentes Verdes/genética , Potenciales Postsinápticos Inhibidores/fisiología , Larva , Terapia por Láser/métodos , Potenciales de la Membrana/fisiología , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Técnicas de Placa-Clamp , Rodaminas/metabolismo , Médula Espinal/fisiología , NataciónRESUMEN
The p2 progenitor domain in the ventral spinal cord gives rise to two interneuron subtypes: V2a and V2b. Delta-Notch-mediated cell-cell interactions between postmitotic immature neurons have been implicated in the segregation of neuron subtypes. However, lineage relationships between V2a and V2b neurons have not been reported. We address this issue using Tg[vsx1:GFP] zebrafish, a model system in which high GFP expression is initiated near the final stage of p2 progenitors. Cell fates were followed in progeny using time-lapse microscopy. Results indicate that the vast majority, if not all, of GFP-labeled p2 progenitors divide once to produce V2a/V2b neuron pairs, indicating that V2a and V2b neurons are generated by the asymmetric division of pair-producing progenitor cells. Together with evidence that Notch signaling is involved in the cell fate specification process, our results strongly suggest that Delta-Notch interactions between sister cells play a crucial role in the final outcome of these asymmetric divisions. This mechanism for determining cell fate is similar to asymmetric divisions that occur during Drosophila neurogenesis, where ganglion mother cells divide once to produce distinct neurons. However, unlike in Drosophila, the divisional axes of p2 progenitors in zebrafish were not fixed. We report that the terminal division of pair-producing progenitor cells in vertebrate neurogenesis can reproducibly produce two distinct neurons through a mechanism that may not depend on the orientation of the division axis.