Volume X-Ray Micro-Computed Tomography Analysis of the Early Cephalized Central Nervous System in a Marine Flatworm, Stylochoplana pusilla.

Platyhelminthes are a phylum of simple bilaterian invertebrates with prototypic body systems. Compared with non-bilaterians such as cnidarians, the bilaterians are likely to exhibit integrated free-moving behaviors, which require a concentrated nervous system "brain" rather than the distributed nervous system of radiatans. Marine flatworms have an early cephalized 'central' nervous system compared not only with non-bilaterians but also with parasitic flatworms or freshwater planarians. In this study, we used the marine flatworm Stylochoplana pusilla as an excellent model organism in Platyhelminthes because of the early cephalized central nervous system. Here, we investigated the three-dimensional structures of the flatworm central nervous system by the use of X-ray micro-computed tomography (micro-CT) in a synchrotron radiation facility. We found that the obtained tomographic images were sufficient to discriminate some characteristic structures of the nervous system, including nerve cords around the cephalic ganglion, mushroom body-like structures, and putative optic nerves forming an optic commissure-like structure. Through the micro-CT imaging, we could obtain undistorted serial section images, permitting us to visualize precise spatial relationships of neuronal subpopulations and nerve tracts. 3-D micro-CT is very effective in the volume analysis of the nervous system at the cellular level; the methodology is straightforward and could be applied to many other non-model organisms.

Diversity and evolution of serotonergic cells in taste buds of elasmobranchs and ancestral actinopterygian fish.

A subset of gustatory cells are serotonin immunoreactive (ir) in the mammalian taste bud. In the taste bud of lamprey, elongated gustatory-like cells are also serotonin-ir. In contrast, flattened serotonin-ir cells are located only in the basal region of the taste buds in the teleosts and amphibians. These serotonin-ir cells are termed as basal cells. To evaluate the evolution and diversity of serotonergic cells in the taste bud of amniote animals, we explored the distribution and morphology of serotonin-ir cells in the taste buds of ancestral actinopterygian fish (spotted gar, sturgeon, Polypterus senegalus) and elasmobranch (stingray). In all examined animals, the taste buds contained serotonin-ir cells in their basal part. The number of serotonin-ir basal cells in each taste bud was different between these fish species. They were highest in the stingray and decreased in the order of the Polypterus, sturgeon, and gar. While serotonin immunoreactivity was observed only in the basal cells in the taste buds of the ancestral actinopterygian fish, some elongated cells were also serotonin-ir in addition to the basal cells in the stingray taste buds. mRNA of tryptophan hydroxylase 1 (tph1), a rate-limiting enzyme of the serotonin synthesis, is expressed in both the elongated and basal cells of stingray taste buds, indicating that these cells synthesize the serotonin by themselves. These results suggest that the serotonin-ir basal cells arose from the ancestor of the cartilaginous fish, and serotonin-ir cells in the elasmobranch taste bud exhibit an intermediate aspect between the lamprey and actinopterygian fish.

Histological and Molecular Characterization of the Inferior Olivary Nucleus and Climbing Fibers in the Goldfish, Carassius auratus.

The cerebellum receives inputs via the climbing fibers originating from the inferior olivary nucleus in the ventral medulla. In mammals, the climbing fibers entwine and terminate onto both major and peripheral branches of dendrites of the Purkinje cells. In this study, the inferior olivary nucleus and climbing fiber in the goldfish were investigated with several histological techniques. By neural tracer application to the hemisphere of the cerebellum, labeled inferior olivary neurons were found in the ventral edge of the contralateral medulla. Kainate stimulated Co + + uptake and gephyrin immunoreactivities were found in inferior olivary neurons, indicating, respectively, that they receive both excitatory (glutamatergic) and inhibitory (GABAergic or glycinergic) inputs. Inferior olivary neurons express vglut2.1 transcripts, suggesting they are glutamatergic. Around 85% of inferior olivary neurons were labeled with anti-calretinin antiserum. Calretinin immunoreactive (ir) climbing fiber terminal-like structures were distributed near the Purkinje cells and in the molecular layer. Double labeling immunofluorescence with anti-calretinin and zebrin II antisera revealed that the calretinin-ir climbing fibers run along and made synaptic-like contacts on the major dendrites of the zebrin II-ir Purkinje cells. In teleost fish, cerebellar efferent neurons, eurydendroid cells, also lie near the Purkinje cells and extend dendrites outward to intermingle with dendrites of the Purkinje cells within the molecular layer. Here we found no contacts between the climbing fiber terminals and the eurydendroid cell dendrites. These results support the idea that Purkinje cells, but not eurydendroid cells, receive strong inputs via the climbing fibers, similar to the mammalian situation.

Identification of adult nephron progenitors capable of kidney regeneration in zebrafish.

Loss of kidney function underlies many renal diseases. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10-30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies.

Distribution, Innervation, and Cellular Organization of Taste Buds in the Sea Catfish, Plotosus japonicus.

The gustatory system of the sea catfish Plotosus japonicus, like that of other catfishes, is highly developed. To clarify the details of the morphology of the peripheral gustatory system of Plotosus, we used whole-mount immunohistochemistry to investigate the distribution and innervation of the taste buds within multiple organs including the barbels, oropharyngeal cavity, fins (pectoral, dorsal, and caudal), and trunk. Labeled taste buds could be observed in all the organs examined. The density of the taste buds was higher along the leading edges of the barbels and fins; this likely increases the chance of detecting food. In all the fins, the taste buds were distributed in linear arrays parallel to the fin rays. Labeling of nerve fibers by anti-acetylated tubulin antibody showed that the taste buds within each sensory field are innervated in different ways. In the barbels, large nerve bundles run along the length of the organ, with fascicles branching off to innervate polygonally organized groups of taste buds. In the fins, nerve bundles run along the axis of fin rays to innervate taste buds lying in a line. In each case, small fascicles of fibers branch from large bundles and terminate within the basal portions of the taste buds. Serotonin immunohistochemistry demonstrated that most of the taste buds in all the organs examined contained disk-shaped serotonin-immunopositive cells in their basal region. This indicates a similar organization of the taste buds, in terms of the existence of serotonin-immunopositive basal cells, across the different sensory fields in this species.

Acetylcholine receptors enable the transport of rapsyn from the Golgi complex to the plasma membrane.

The accumulation of acetylcholine receptors (AChRs) at nerve terminals is critical for signal transmission at the neuromuscular junction, and rapsyn is essential for this process. Previous studies suggest that AChRs might direct rapsyn self-clusters to the synapse. In vivo experiments with fluorescently tagged AChR or rapsyn in zebrafish larvae revealed that rapsyn self-clusters separate from AChRs did not exist before synapse formation. Examination of rapsyn in the AChR-less mutant sofa potato revealed that rapsyn in the absence of AChR was localized in the Golgi complex. Expression of muscle-type AChR in sofa potato restored synaptic clustering of rapsyn, while neuronal type AChR had no effect. To determine whether this requirement of protein interaction is reciprocal, we examined the mutant twitch once, which has a missense mutation in rapsyn. While the AChRs distributed nonsynaptically on the plasma membrane in twitch once, mutant rapsyn was retained in the Golgi complex. We conclude that AChRs enable the transport of rapsyn from the Golgi complex to the plasma membrane through a molecule-specific interaction.

Transgenic line with gal4 insertion useful to study morphogenesis of craniofacial perichondrium, vascular endothelium-associated cells, floor plate, and dorsal midline radial glia during zebrafish development.

Zebrafish is a good model for studying vertebrate development because of the availability of powerful genetic tools. We are interested in the study of the craniofacial skeletal structure of the zebrafish. For this purpose, we performed a gene trap screen and identified a Gal4 gene trap line, SAGFF(LF)134A. We then analyzed the expression pattern of SAGFF(LF)134A;Tg(UAS:GFP) and found that green fluorescent protein (GFP) was expressed not only in craniofacial skeletal elements but also in the vascular system, as well as in the nervous system. In craniofacial skeletal elements, strong GFP expression was detected not only in chondrocytes but also in the perichondrium. In the vascular system, GFP was expressed in endothelium-associated cells. In the spinal cord, strong GFP expression was found in the floor plate, and later in the dorsal radial glia located on the midline. Taking advantage of this transgenic line, which drives Gal4 expression in specific tissues, we crossed SAGFF(LF)134A with several UAS reporter lines. In particular, time-lapse imaging of photoconverted floor-plate cells of SAGFF(LF)134A;Tg(UAS:KikGR) revealed that the floor-plate cells changed their shape within 36 h from cuboidal/trapezoidal to wine glass shaped. Moreover, we identified a novel mode of association between axons and glia. The putative paths for the commissural axons, including pax8-positive CoBL interneurons, were identified as small openings in the basal endfoot of each floor plate. Our results indicate that the transgenic line would be useful for studying the morphogenesis of less-well-characterized tissues of interest, including the perichondrium, dorsal midline radial glia, late-stage floor plate, and vascular endothelium-associated cells.

Morphological analysis of the cerebellum and its efferent system in a basal actinopterygian fish, Polypterus senegalus.

Although all vertebrate cerebella contain granule cells, Purkinje cells, and efferent neurons, the cellular arrangement and neural circuitry are highly diverse. In amniotes, cerebellar efferent neurons form clusters, deep cerebellar nuclei, lie deep in the cerebellum, and receive synaptic inputs from Purkinje cells but not granule cells. However, in the cerebellum of teleosts, the efferent neurons, called eurydendroid cells, lie near the cell bodies of Purkinje cells and receive inputs both from axons of Purkinje cells and granule cell parallel fibers. It is largely unknown how the cerebellar structure evolved in ray-finned fish (actinopterygians). To address this issue, we analyzed the cerebellum of a bichir Polypterus senegalus, one of the most basal actinopterygians. We found that the cell bodies of Purkinje cells are not aligned in a layer; incoming climbing fibers terminate mainly on the basal portion of Purkinje cells, revealing that the Polypterus cerebellum has unique features among vertebrate cerebella. Retrograde labeling and marker analyses of the efferent neurons revealed that their cell bodies lie in restricted granular areas but not as deep cerebellar nuclei in the cerebellar white matter. The efferent neurons have long dendrites like eurydendroid cells, although they do not reach the molecular layer. Our findings suggest that the efferent system of the bichir cerebellum has intermediate features between teleosts and amniote vertebrates, and provides a model to understand the basis generating diversity in actinopterygian cerebella.

A modified acetylcholine receptor delta-subunit enables a null mutant to survive beyond sexual maturation.

The contraction of skeletal muscle is dependent on synaptic transmission through acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). The lack of an AChR subunit causes a fetal akinesia in humans, leading to death in the first trimester and characteristic features of Fetal Akinesia Deformation Sequences (FADS). A corresponding null mutation of the delta-subunit in zebrafish (sofa potato; sop) leads to the death of embryos around 5 d postfertilization (dpf). In sop(-/-) mutants, we expressed modified delta-subunits, with one (delta1YFP) or two yellow fluorescent protein (delta2YFP) molecules fused at the intracellular loop, under the control of an alpha-actin promoter. AChRs containing these fusion proteins are fluorescent, assemble on the plasma membrane, make clusters under motor neuron endings, and generate synaptic current. We screened for germ-line transmission of the transgene and established a line of sop(-/-) fish stably expressing the delta2YFP. These delta2YFP/sop(-/-) embryos can mount escape behavior close to that of their wild-type siblings. Synaptic currents in these embryos had a smaller amplitude, slower rise time, and slower decay when compared with wild-type fish. Remarkably, these embryos grow to adulthood and display complex behaviors such as feeding and breeding. To the best of our knowledge, this is the first case of a mutant animal corresponding to first trimester lethality in human that has been rescued by a transgene and survived to adulthood. In the rescued fish, a foreign promoter drove the transgene expression and the NMJ had altered synaptic strength. The survival of the transgenic animal delineates requirements for gene therapies of NMJ.

A lambda-shaped retractor lentis muscle in the yellowfin goby Acanthogobius flavimanus.

We identified a morphologically uncommon piscine retractor lentis muscle in the yellowfin goby Acanthogobius flavimanus. This lentis muscle has a shape similar to the Greek small letter lambda (λ). The two legs of the muscle are attached to the retinal periphery at the ventral eyecup, while the tip is connected to the lens surface by a ligament. Scanning electron microscopy showed that the fibers of the lentis muscle run along the length of both the anterior and posterior legs. Immunolabeling with antiacetylated tubulin antibody and neuronal tracing with DiI of the whole lentis muscle revealed that the anterior leg is innervated by one or more nerves. The topographic distribution of ganglion cells in the retina was investigated to identify the visual axis. Three high cell density areas were observed in the dorso-temporal, ventro-nasals and ventro-temporal retina. These findings suggest that the λ-shaped lentis muscle may enable accommodatory movement of the lens toward the temporal as well as the nasal and/or ventral retina.

Imaging mass spectrometry of gold nanoparticles in a tissue section as an immunohistochemical staining mass probe.

For analysis of low abundance peptides in a tissue section, immunohistochemical staining through antibody-antigen interaction is a usual technique. The antibody is conjugated with a probe moiety that aids in highly sensitive detection. Gold nanoparticles, which show excellent chemical stability and variation of surface modifications, are expected to act as a sensitive mass probe to desorb gold ions (Au+ , Au2 + , Au3 + ) that are distinguishable from fragment ions from organic molecules. Here, green fluorescent proteins (GFP) in a tissue section of a transgenic zebrafish were detected by the gold mass probe conjugated with antibodies. Due to the efficient ionization and desorption of gold ions, imaging mass spectrometry of Au2 + ions indicated the distribution of gold nanoparticles stained in a tissue section, and the mass signal distribution was consistent with the area where the GFP-expressing cells were distributed. Conventional immunofluorescence techniques showed intense autofluorescence that come from intrinsic fluorophores in the tissue section. In contrast, the gold nanoparticles acted as an immunostaining mass probe that displayed significantly lower background signals.

Calcium-fluxing glutamate receptors associated with primary gustatory afferent terminals in goldfish (Carassius auratus).

Presynaptic ionotropic glutamate receptors modulate transmission at primary afferent synapses in several glutamatergic systems. To test whether primary gustatory afferent fibers express Ca(2+)-permeable AMPA/kainate receptors, we utilized kainate-stimulated uptake of Co(2+) along with immunocytochemistry for the Ca(2+)-binding proteins (CaBPs) calbindin and calretinin to investigate the primary gustatory afferents in goldfish (Carassius auratus). In goldfish, the primary gustatory nucleus (equivalent to the gustatory portion of the nucleus of the solitary tract) includes the vagal lobe, which is a large, laminated structure protruding dorsally from the medulla. Kainate-stimulated uptake of Co(2+) (a measure of Ca(2+)-fluxing glutamate receptors) shows punctate staining distributed in the distinct laminar pattern matching the layers of termination of the primary gustatory afferent fibers. In addition, CaBP immunocytochemistry, which correlates highly with expression of Ca(2+)-permeable AMPA/kainate receptors, shows a laminar pattern of distribution similar to that found with kainate-stimulated cobalt uptake. Nearly all neurons of the vagal gustatory ganglion show Co(2+) uptake and are immunopositive for CaBPs. Transection of the vagus nerve proximal to the ganglion results in loss of such punctate Co(2+) uptake and of punctate CaBP staining as soon as 4 days postlesion. These results are consonant with the presence of Ca(2+)-fluxing glutamate receptors on the presynaptic terminals of primary gustatory terminals, providing an avenue for modulation of primary gustatory input.

Central mechanisms underlying fish swimming.

Although the basic swimming rhythm is created by central pattern generators (CPGs) located in each spinal segment, command signals from the brain should be indispensable for the activation of CPGs to initiate swimming. We hypothesized that the nucleus of medial longitudinal fascicles (Nflm) is the midbrain locomotor region driving swimming rhythms in teleosts. To test this hypothesis, we recorded neuronal activities from Nflm neurons in swimming carp and analyzed the cytoarchitecture of the nucleus. We identified two types of Nflm neurons exhibiting electric activities closely related to swimming rhythms. Remarkably, tonic neurons that continued firing during swimming were found. The Nflm and neighboring oculomotor nucleus contain about 600 neurons in total, and among them as many as 500 were labeled retrogradely by an intraspinal tracer implantation and 400 neurons showed glutamatergic immunoreactivity. They are the most likely candidates for the descending neurons as the origin of driving signals that initiate swimming. Double-labeling experiments demonstrated direct connections of Nflm neurons to spinal neurons consisting of the CPG. These data imply that most Nflm neurons possibly exert an excitatory drive to the spinal CPGs through the descending axons with excitatory transmitter(s), probably glutamate. Furthermore, we confirmed that the caudal part of Nflm and the rostral part of the oculomotor nucleus overlap rostrocaudally by approximately 200 mum. In connection with the control of swimming by the brain, we carried out experiments to clarify the efferent system of the cerebellum of the goldfish. Cerebellar efferent fibers terminated in most brain regions except for the telencephalon. Importantly, the cerebellum projected also to the Nflm, suggesting the involvement of this brain region in the control of swimming. We have also determined that in the carp so-called eurydendroid cells are cerebellar efferent neurons.

Suppression of Neu1 sialidase delays the absorption of yolk sac in medaka (Oryzias latipes) accompanied with the accumulation of α2-3 sialo-glycoproteins.

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Recently, medaka sialidase Neu1 has been cloned and its enzymatic properties were investigated. Although enzymatic properties of this sialidase, such as optimal pH and substrate specificity, exhibits high similarity with human NEU1, Neu1 physiological functions in fish are still unclear. Here, to understand Neu1 significance in medaka embryogenesis, sialidase translation knockdown was carried out with one-cell stage fertilized egg using morpholino oligo injection. Neu1 exhibited desialylation of α2-3 sialic acid linkage in vitro and lysosomal localization in medaka caudal fin primary cells. Chloroquine treatment, inhibitor of lysosomal enzymes, caused an accumulation of α2-3 sialo-glycoproteins in the primary cells. During the embryogenesis neu1 mRNA level was elevated until 3.5 day post fertilization (dpf) while an initial decrease of α2-3 sialo-glycoprotein was observed around the same developmental stage. Neu1 knockdown by morpholino oligo induced some abnormal phenotypes such as delay of yolk sac absorption and small embryos. Sialidase-knockdown embryos also showed increase of heart rate in 5.5 and 6.5 dpf. Furthermore, about 37% decrease of hatching rate was observed in Neu1-MO treated embryos compared with control MO. Embryos showing severe phenotypes stopped embryogenesis at the late stage of development. Alteration of embryonic sialo-glycoproteins induced by morpholino injection was examined by lectin blotting to clarify the mechanism of abnormal development. As a result, degradation of several α2-3 sialo-glycoproteins was suppressed in Neu1-MO embryo, possibly induced by the interruption of lysosomal desialylation toward yolk glycoprotein. Our results suggest that medaka Neu1 could be crucial for embryonic development through the degradation of yolk sac nutrition.

Co-occurrence of calcium-binding proteins and calcium-permeable glutamate receptors in the primary gustatory nucleus of goldfish.

Primary vagal gustatory afferents utilize glutamate as a neurotransmitter acting on AMPA/kainate receptors of second-order neurons. Some forms of ionotropic glutamate receptors permit passage of Ca++ ions upon activation by appropriate ligands. Calcium-binding proteins (CaBPs) play a buffering role for regulating the concentration of intracellular calcium. In the present study, we used immunohistochemistry to examine the distribution and morphology of neurons with CaBPs, including calretinin, calbindin, and parvalbumin, and to compare this distribution with neurons exhibiting Ca++-permeable glutamate receptors as determined by kainate-stimulated uptake of Co++ in the vagal lobe of goldfish. Calretinin- and calbindin-positive neurons occurred throughout the sensory zone including round unipolar, horizontal; and perpendicular bipolar or multipolar somata. Parvalbumin neurons were mainly round monopolar neurons, especially common in the superficial layers of the sensory zone. In the motor zone, while parvalbumin labeled nearly all motoneurons, calretinin labeled only external motoneurons. In double labeling with calretinin and parvalbumin, few neurons in the sensory layer labeled with both antisera. Immunocytochemistry following kainate-stimulate uptake of Co++ showed that most calretinin, but few parvalbumin immunopositive neurons also were labeled by cobalt in the central and deep layers of the sensory zone. All motoneurons were labeled by Co++, including those immunoreactive for calretinin or parvalbumin. These results indicate that calretinin expression is strongly correlated with calcium-permeable ionotropic glutamate receptors in the neurons of the sensory zone of the goldfish vagal lobe, but even within this limited region, not all Ca++-permeable neurons possess any of the CaBPs examined.

Morphology and immunohistochemistry of efferent neurons of the goldfish corpus cerebelli.

In teleosts, cerebellar efferent neurons, known as eurydendroid cells, are dispersed within the cerebellar cortex rather than coalescing into deep cerebellar nuclei. To clarify their morphology, eurydendroid cells were labeled retrogradely by biotinylated dextran amine injection into the base of the corpus cerebelli. Labeling allowed the cells to be classified into three types-fusiform, polygonal, and monopolar-depending on their somal shapes and numbers of primary dendrites. The fusiform and polygonal type cells were distributed not only in the Purkinje cell layer but also in the molecular and granule cell layers. The monopolar type cells were distributed predominantly in the Purkinje cell layer of the ventrocaudal portion of the corpus cerebelli. These results suggest that there are some functional differences between these eurydendroid cell types. The eurydendroid cells were double-labeled by retrograde labeling and immunohistochemistry using specific antibodies against GABA, aspartate, and zebrin II. No GABA-like immunoreactivity was detected in the retrogradely labeled eurydendroid cells. About half of retrogradely labeled cells were immunoreactive to the anti-aspartate antibody, suggesting that some eurydendroid cells utilize aspartate as a neurotransmitter. Zebrin II reacts with cerebellar Purkinje cells but left all retrogradely labeled neurons nonreactive, although some of these were surrounded by immunopositive fibers. This relationship between the eurydendroid and Purkinje cells is similar to that between the deep cerebellar nuclei and Purkinje cells in mammals.

Partial cloning and expression of mRNA coding choline acetyltransferase in the spinal cord of the goldfish, Carassius auratus.

Choline acetyltransferase (ChAT, EC 2.3.1.6) synthesizes a neurotransmitter, acetylcholine in cholinergic neurons. ChAT is considered to be the most specific marker for cholinergic neurons. To obtain a better marker of the neurons, as the first step, we isolated a partial ChAT cDNA from the goldfish (Carassius auratus) brain by RT-PCR methods. The partial cDNA of the goldfish ChAT was composed of 718 nucleotides. The amino acid sequence of the goldfish ChAT is approximately 70% identical to those of mammalian and chicken ChAT. Northern blot analysis demonstrated that ChAT mRNA was expressed in the brain and the spinal cord of the goldfish, and much abundant in the spinal cord. In the spinal cord of the goldfish, ChAT-positive neurons were detected mainly in the ventral horn by in situ hybridization. In addition, fluorescence in situ hybridization combined with a retrograde labeling by using True Blue demonstrated ChAT mRNA positive neurons were exactly motoneurons. In the cord, putative presynaptic sympathetic neurons were also labeled.

Betanodavirus as a novel transneuronal tracer for fish.

In order to obtain a potential new tool to analyze networks of the central nervous system of teleost fishes, we tested a fish-pathogenic betanodavirus, sevenband grouper nervous necrosis virus (SGNNV), as a transneuronal tracer using the freshwater angelfish Pterophyllum scalare as a test animal. Intravitreous injections of SGNNV into the right eye resulted first in the labelings of neuronal cell bodies in the ganglion cell layers of the retina and then those in the inner and outer nuclear layers in sequence. For the first time, labeled neurons were found also in the stratum periventriculare of the contralateral optic tectum, the ventrolateral and ventromedial thalamic nuclei, and the periventricular nucleus of posterior tuberculum in the brain, then the periventricular pretectal nucleus pars dorsalis and pars ventralis. In contrast, by injections of biotinylated dextran amine into the eye no labeled cell bodies were observed in these brain areas, but axons and terminals were labeled anterogradely. These results suggest that the virus could be transported in both directions in axons of the first order neuron and transfected the second and third order neurons by passing across synaptic clefts, and that this technique is practically applicable to the study of neurobiology in teleost.

Identification of initially appearing glycine-immunoreactive neurons in the embryonic zebrafish brain.

Glycine is a major inhibitory neurotransmitter in the central nervous system of vertebrates. Here, we report the initial development of glycine-immunoreactive (Gly-ir) neurons and fibers in zebrafish. The earliest Gly-ir cells were found in the hindbrain and rostral spinal cord by 20 h post-fertilization (hpf). Gly-ir cells in rhombomeres 5 and 6 that also expressed glycine transporter 2 (glyt2) mRNA were highly stereotyped; they were bilaterally located and their axons ran across the midline and gradually turned caudally, joining the medial longitudinal fascicles in the spinal cord by 24 hpf. Gly-ir neurons in rhombomere 5 were uniquely identified, since there was one per hemisegment, whereas the number of Gly-ir neurons in rhombomere 6 were variable from one to three per hemisegment. Labeling of these neurons by single-cell electroporation and tracing them until the larval stage revealed that they became MiD2cm and MiD3cm, respectively. The retrograde labeling of reticulo-spinal neurons in Tg(glyt2:gfp) larva, which express GFP in Gly-ir cells, and a genetic mosaic analysis with glyt2:gfp DNA construct also supported this notion. Gly-ir cells were also distributed widely in the anterior brain by 27 hpf, whereas glyt2 was hardly expressed. Double staining with anti-glycine and anti-GABA antibodies demonstrated distinct distributions of Gly-ir and GABA-ir cells, as well as the presence of doubly immunoreactive cells in the brain and placodes. These results provide evidence of identifiable glycinergic (Gly-ir/glyt2-positive) neurons in vertebrate embryos, and they can be used in further studies of the neurons' development and function at the single-cell level.

Formation of the spinal network in zebrafish determined by domain-specific pax genes.

In the formation of the spinal network, various transcription factors interact to develop specific cell types. By using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) was inserted into the pax8 gene. RFP insertion marked putative pax8-lineage cells with fluorescence and inhibited pax8 expression in homozygous embryos. Pax8 homozygous embryos displayed defects in the otic vesicle, as previously reported in studies with morpholinos. The pax8 homozygous embryos survived to adulthood, in contrast to mammalian counterparts that die prematurely. RFP is expressed in the dorsal spinal cord. Examination of the axon morphology revealed that RFP(+) neurons include commissural bifurcating longitudinal (CoBL) interneurons, but other inhibitory neurons such as commissural local (CoLo) interneurons and circumferential ascending (CiA) interneurons do not express RFP. We examined the effect of inhibiting pax2a/pax8 expression on interneuron development. In pax8 homozygous fish, the RFP(+) cells underwent differentiation similar to that of pax8 heterozygous fish, and the swimming behavior remained intact. In contrast, the RFP(+) cells of pax2a/pax8 double mutants displayed altered cell fates. CoBLs were not observed. Instead, RFP(+) cells exhibited axons descending ipsilaterally, a morphology resembling that of V2a/V2b interneurons.