Expression patterns of Sema3A in developing amniote limbs: With reference to the diversification of peripheral nerve innervation.

Paired limbs were acquired in the ancestor of tetrapods and their morphology has been highly diversified in amniotes in relation to the adaptive radiation to the terrestrial environment. These morphological changes may have been induced by modification of the developmental program of the skeletal or muscular system. To complete limb modification, it is also important to change the neuronal framework, because the functions of the limbs rely on neural circuits that involve coordinated movement. Previous studies have shown that class 3 semaphorins (Sema3 semaphorins), which act as repulsive axonal guidance cues, play a crucial role in the formation of the peripheral nerves in mice. Here, we studied the expression pattern of Sema3A orthologues in embryos of developing amniotes, including mouse, chick, soft-shelled turtle, and ocelot gecko. Sema3A transcripts were expressed in restricted mesenchymal parts of the developing limb primordium in all animals studied, and developing spinal nerves appeared to extend through Sema3A-negative regions. These results suggest that a Sema3A-dependent guidance system plays a key role in neuronal circuit formation in amniote limbs. We also found that Sema3A partially overlapped with the distribution of cartilage precursor cells. Based on these results, we propose a model in which axon guidance and skeletogenesis are linked by Sema3A; such mechanisms may underlie functional neuron rearrangement during limb diversification.

Development of the thalamo-dorsal ventricular ridge tract in the Chinese soft-shelled turtle, Pelodiscus sinensis.

With the exception of that from the olfactory system, the vertebrate sensory information is relayed by the dorsal thalamus (dTh) to be carried to the telencephalon via the thalamo-telencephalic tract. Although the trajectory of the tract from the dTh to the basal telencephalon seems to be highly conserved among amniotes, the axonal terminals vary in each group. In mammals, thalamic axons project onto the neocortex, whereas they project onto the dorsal pallium and the dorsal ventricular ridge (DVR) in reptiles and birds. To ascertain the evolutionary development of the thalamo-telencephalic connection in amniotes, we focused on reptiles. Using the Chinese soft-shelled turtle (Pelodiscus sinensis), we studied the developmental course of the thalamic axons projecting onto the DVR. We found, during the developmental period when the thalamo-DVR connection forms, that transcripts of axon guidance molecules, including EphA4 and Slit2, were expressed in the diencephalon, similar to the mouse embryo. These results suggest that the basic mechanisms responsible for the formation of the thalamo-telencephalic tract are shared across amniote lineages. Conversely, there was a characteristic difference in the expression patterns of Slit2, Netrin1, and EphrinA5 in the telencephalon between synapsid (mammalian) and diapsid (reptilian and avian) lineages. This indicates that changes in the expression domains of axon guidance molecules may modify the thalamic axon projection and lead to the diversity of neuronal circuits in amniotes.

Reptiles: a new model for brain evo-devo research.

Vertebrate brains exhibit vast amounts of anatomical diversity. In particular, the elaborate and complex nervous system of amniotes is correlated with the size of their behavioral repertoire. However, the evolutionary mechanisms underlying species-specific brain morphogenesis remain elusive. In this review we introduce reptiles as a new model organism for understanding brain evolution. These animal groups inherited ancestral traits of brain architectures. We will describe several unique aspects of the reptilian nervous system with a special focus on the telencephalon, and discuss the genetic mechanisms underlying reptile-specific brain morphology. The establishment of experimental evo-devo approaches to studying reptiles will help to shed light on the origin of the amniote brains.

Afferent and efferent connections of the nucleus prethalamicus in the yellowfin goby Acanthogobius flavimanus.

The nucleus prethalamicus (PTh) receives fibers from the optic tectum and then projects to the dorsal telencephalon in the yellowfin goby Acanthogobius flavimanus. However, it remained unclear whether the PTh is a visual relay nucleus, because the optic tectum receives not only visual but also other sensory modalities. Furthermore, precise telencephalic regions receiving prethalamic input remained unknown in the goby. We therefore investigated the full set of afferent and efferent connections of the PTh by direct tracer injections into the nucleus. Injections into the PTh labeled cells in the optic tectum, ventromedial thalamic nucleus, central and medial parts of the dorsal telencephalon, and caudal lobe of the cerebellum. We found that the somata of most tecto-prethalamic neurons are present in the stratum periventriculare. Their dendrites ascend to reach the major retinorecipient layers of the tectum. The PTh is composed of two subnuclei (medial and lateral) and topographic organization was appreciated only for tectal projections to the lateral subnucleus (PTh-l), which also receives sparse retinal projections. In contrast, the medial subnucleus receives fibers only from the medial tectum. We found that the PTh projects to nine subregions in the dorsal telencephalon and four in the ventral telencephalon. Furthermore, cerebellar injections revealed that cerebello-prethalamic fibers cross the midline twice to innervate the PTh-l on both sides. The present study is the first detailed report on the full set of the connections of PTh, which suggests that the PTh relays visual information from the optic tectum to the telencephalon.

FGF- and SHH-based molecular signals regulate barbel and craniofacial development in catfish.

BACKGROUND: Catfish (Siluriformes) are characterized by unique morphologies, including enlarged jaws with movable barbels and taste buds covering the entire body surface. Evolution of these characteristics was a crucial step in their adaptive radiation to freshwater environments. However, the developmental processes of the catfish craniofacial region and taste buds remain to be elucidated; moreover, little is known about the molecular mechanisms underlying the morphogenesis of these structures. RESULTS: In Amur catfish (Silurus asotus), three pairs of barbel primordia are formed by 2 days post-fertilization (dpf). Innervation of the peripheral nerves and formation of muscle precursors are also established during early development. Taste buds from the oral region to the body trunk are formed by 4 dpf. We then isolated catfish cognates Shh (SaShh) and Fgf8 (SaFgf8), which are expressed in maxillary barbel primordium at 1-2 dpf. Further, SHH signal inhibition induces reduction of mandibular barbels with abnormal morphology of skeletal elements, whereas it causes no apparent abnormality in the trigeminal and facial nerve morphology. We also found that mandibular barbel lengths and number of taste buds are reduced by FGF inhibition, as seen in SHH signal inhibition. However, unlike with SHH inhibition, the abnormal morphology of the trigeminal and facial nerves was observed in FGF signal-inhibited embryos. CONCLUSION: The developmental processes of Amur catfish are consistent with those reported for other catfish species. Thus, developmental aspects of craniofacial structures and taste buds may be conserved in Siluriformes. Our findings also suggest that SHH signaling plays a crucial role in the formation of barbels and taste buds, without affecting nerve projection, while FGF signaling is required for the development of barbels, taste buds, and branchial nerves. Thus, SHH and FGF signaling plays key roles in the ontogenesis and evolution of some catfish-specific characteristics.

Atlas of the telencephalon based on cytoarchitecture, neurochemical markers, and gene expressions in Rhinogobius flumineus [Mizuno, 1960].

Gobiida is a basal subseries of percomorphs in teleost fishes, holding a useful position for comparisons with other orders of Percomorpha as well as other cohort of teleosts. Here, we describe a telencephalic atlas of a Gobiida species Rhinogobius flumineus (Mizuno, Memoirs of the College of Science, University of Kyoto, Series B: Biology, 1960; 27, 3), based on cytoarchitectural observations, combined with analyses of the distribution patterns of neurochemical markers and transcription factors. The telencephalon of R. flumineus shows a number of features distinct from those of other teleosts. Among others, the followings were of special note. (a) The lateral part of dorsal telencephalon (Dl), which is known as a visual center in other teleosts, is composed of as many as seven regions, some of which are conspicuous, circumscribed by cell plates. These subdivisions of the Dl can be differentiated clearly by differential soma size and color with Nissl-staining, and distribution patterns of neural markers. (b) Cell populations continuous with the ventral region of dorsal part of ventral telencephalon (vVd) exhibit extensive dimension. Especially, portion 1 of the central part of ventral telencephalon appears to represent a cell population laterally translocated from the vVd, forming a large cluster of small cells that penetrate deep into the central part of dorsal telencephalon. (c) The magnocellular subdivision of dorsal part of dorsal telencephalon (Ddmg) contains not only large cells but also vglut2a-positive clusters of small cells that cover a wide range of the caudal Ddmg. Such clusters of small cells have not been observed in the Ddmg of other teleosts.

Nervous system disruption and swimming abnormality in early-hatched pufferfish (Takifugu niphobles) larvae caused by pyrene is independent of aryl hydrocarbon receptors.

Pyrene, a member of the polycyclic aromatic hydrocarbons (PAHs), contributes to abnormality in the size of the brain and the swimming behavior of pufferfish (Takifugu niphobles) larvae. We hypothesized that the aryl hydrocarbon receptor (AHR) may mediate pyrene-induced toxic effects because AHR is assumed to be a candidate for the downstream target of PAHs in many cases. To identify the contribution of AHR on developing pufferfish, we performed exposure experiments using ß-naphthoflavone, an agonist of AHR. We found that the toxic effects of pyrene and ß-naphthoflavone in pufferfish larvae are fundamentally different. Pyrene specifically induced problems in the developing midbrain and in swimming behavior, while ß-naphthoflavone affected the heartbeat rate and the size of the yolk. These results suggest that the behavioral and morphological abnormality caused by pyrene exposure is mediated by an AHR-independent pathway. Alternatively, defects caused by pyrene may be attributed to the inhibition of the FGF signal.

An Evolutionary Hypothesis of Binary Opposition in Functional Incompatibility about Habenular Asymmetry in Vertebrates.

Many vertebrates have asymmetrical circuits in the nervous system. There are two types of circuit asymmetry. Asymmetrical circuits in sensory and/or motor systems are usually related to lateralized behaviors. It has been hypothesized that spatial asymmetry in the environment and/or social interactions has led to the evolution of asymmetrical circuits by natural selection. There are also asymmetrical circuits that are not related to lateralized behaviors. These circuits lie outside of the sensory and motor systems. A typical example is found in the habenula (Hb), which has long been known to be asymmetrical in many vertebrates, but has no remarkable relationship to lateralized behaviors. Instead, the Hb is a hub wherein information conveyed to the unilateral Hb is relayed to diverging bilateral nuclei, which is unlikely to lead to lateralized behavior. Until now, there has been no hypothesis regarding the evolution of Hb asymmetry. Here, we propose a new hypothesis that binary opposition in functional incompatibility applies selection pressure on the habenular circuit and leads to asymmetry. Segregation of the incompatible functions on either side of the habenula is likely to enhance information processing ability via creating shorter circuits and reducing the cost of circuit duplication, resulting in benefits for survival. In zebrafish and mice, different evolutionary strategies are thought to be involved in Hb asymmetry. In zebrafish, which use a strategy of structurally fixed asymmetry, the asymmetrical dorsal Hb leads to constant behavioral choices in binary opposition. In contrast, in mice, which use a strategy of functionally flexible lateralization, the symmetrical lateral Hb is functionally lateralized. This makes it possible to process complicated information and to come to variable behavioral choices, depending on the specific situation. These strategies are thought to be selected for and preserved by evolution under selection pressures of rigidity and flexibility of sociability in zebrafish and mice, respectively, as they are beneficial for survival. This hypothesis is highly valuable because it explains how the Hb evolved differently in terms of asymmetry and lateralization among different species. In addition, one can propose possible experiments for the verification of this hypothesis in future research.

Characterization of wheat germ agglutinin ligand on soluble glycoproteins in Caenorhabditis elegans.

Some mutants of Caenorhabditis elegans show altered patterns of ectopic binding with wheat germ agglutinin (WGA). Some of these mutants also have defects of morphogenesis and movement during development. To clarify the structures of WGA-ligands in C. elegans that may be involved in developmental events, we have analyzed glycan structures capable of binding WGA. We isolated glycoproteins from wild-type C. elegans by WGA-affinity chromatography, and analyzed their glycan structures by a combination of hydrazine degradation and fluorescent labeling. The glycoproteins had oligomannose-type and complex-type N-glycans that included agalacto-biantenna and agalacto-tetraantenna glycans. Although the complex-type glycans carried beta-GlcNAc residues at their non-reducing ends, they did not bind to the WGA-agarose-resin. Thus, it was suggested that these N-glycans were not responsible for WGA-binding of the isolated glycoproteins. Hydrazinolysis of the glycoproteins also released a considerable amount of GalNAc monosaccharide. It was surmised that N-acetylgalactosamine was derived from mucin-type O-glycans with the Tn-antigen structure (GalNAcalpha1-O-Ser/Thr). WGA-blotting assay of neoglycoproteins revealed that a cluster of Tn-antigens was a good ligand for WGA. These results suggested that the WGA-ligand in C. elegans is a cluster of alpha-GalNAc monosaccharides linked to mucin-like glycoprotein(s). The observations reported in this paper emphasize the possible significance of mucin-type O-glycans in the development of a multicellular organism.

Lateralization, maturation, and anteroposterior topography in the lateral habenula revealed by ZIF268/EGR1 immunoreactivity and labeling history of neuronal activity.

We report habenular lateralization in a simple transgenic mouse model used for labeling a facet of neuronal activity history. A transgenic construct comprised of a zif268/egr1 immediate-early gene promoter and a gene for normal Venus fluorescent protein with a membrane tag converted promoter activity into long-life fluorescent proteins, which was thought to describe a facet of neuronal activity history by summing neuronal activity. In addition to mapping the immediate-early gene-immunopositive cells, this method helped demonstrate the functionality of the lateral habenular nucleus (LHb). During postnatal development, the LHb was activated between postnatal days 10 and 16. The water-immersion restraint stress also activated the LHb over a similar period. LHb activation was functionally lateralized, but had no directional bias at the population level. Moreover, the posterior LHb was activated in the early stage after the stress, while the anterior LHb was activated in the later stage. Our results indicate lateralization, maturation, and anteroposterior topography of the LHb during postnatal development and the stress response.

Involvement of Slit-Robo signaling in the development of the posterior commissure and concomitant swimming behavior in Xenopus laevis.

INTRODUCTION: During vertebrate development, the central nervous system (CNS) has stereotyped neuronal tracts (scaffolds) that include longitudinal and commissural axonal bundles, such as the medial longitudinal fascicle or the posterior commissure (PC). As these early tracts appear to guide later-developing neurons, they are thought to provide the basic framework of vertebrate neuronal circuitry. The proper construction of these neuronal circuits is thought to be a crucial step for eliciting coordinated behaviors, as these circuits transmit sensory information to the integrative center, which produces motor commands for the effective apparatus. However, the developmental plan underlying some commissures and the evolutionary transitions they have undergone remain to be elucidated. Little is known about the role of axon guidance molecules in the elicitation of early-hatched larval behavior as well. RESULTS: Here, we report the developmentally regulated expression pattern of axon-guidance molecules Slit2 ligand and Robo2 receptor in Xenopus laevis and show that treatment of X. laevis larvae with a slit2- or robo2-morpholino resulted in abnormal swimming behavior. We also observed an abnormal morphology of the PC, which is part of the early axonal scaffold. CONCLUSION: Our present findings suggest that expression patterns of Slit2 and Robo2 are conserved in tetrapods, and that their signaling contributes to the construction of the PC in Xenopus. Given that the PC also includes several types of neurons stemming from various parts of the CNS, it may represent a candidate prerequisite neuronal tract in the construction of subsequent complex neuronal circuits that trigger coordinated behavior.

Structural analysis of N-linked glycans in Caenorhabditis elegans.

Caenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Man(alpha)1-6(Man(alpha)1-3)Man(beta)1-4GlcNAc(beta)1-4GlcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.

Pyrene induces a reduction in midbrain size and abnormal swimming behavior in early-hatched pufferfish larvae.

Spills of heavy oil (HO) have an adverse effect on marine life. We have demonstrated previously that exposure to HO by fertilized eggs of the pufferfish (Takifugu rubripes) induces neural disruption and behavioral abnormality in early-hatched larvae. Here, two kinds of polycyclic aromatic hydrocarbons, pyrene and phenanthrene, were selected to examine their toxic effects on larval behavior of another pufferfish species (T. niphobles). Larvae exposed to pyrene or phenanthrene exhibited no abnormalities in morphology. However, those exposed to pyrene but not phenanthrene swam in an uncoordinated manner, although their swimming distance and speed were normal. The optic tectum, a part of the midbrain, of pyrene-exposed larvae did not grow to full size. Thus, these findings are indicated that pyrene might be a contributor to the behavioral and neuro-developmental toxicity, although there is no indication that it is the only compound participating in the toxicity of the heavy oil mixture.

Developmental epigenetic modification regulates stochastic expression of clustered protocadherin genes, generating single neuron diversity.

In the brain, enormous numbers of neurons have functional individuality and distinct circuit specificities. Clustered Protocadherins (Pcdhs), diversified cell-surface proteins, are stochastically expressed by alternative promoter choice and affect dendritic arborization in individual neurons. Here we found that the Pcdh promoters are differentially methylated by the de novo DNA methyltransferase Dnmt3b during early embryogenesis. To determine this methylation's role in neurons, we produced chimeric mice from Dnmt3b-deficient induced pluripotent stem cells (iPSCs). Single-cell expression analysis revealed that individual Dnmt3b-deficient Purkinje cells expressed increased numbers of Pcdh isoforms; in vivo, they exhibited abnormal dendritic arborization. These results indicate that DNA methylation by Dnmt3b at early embryonic stages regulates the probability of expression for the stochastically expressed Pcdh isoforms. They also suggest a mechanism for a rare human recessive disease, the ICF (Immunodeficiency, Centromere instability, and Facial anomalies) syndrome, which is caused by Dnmt3b mutations.

Development of the dorsal ramus of the spinal nerve in the mouse embryo: involvement of semaphorin 3A in dorsal muscle innervation.

The spinal nerve, which is composed of dorsal root ganglion (DRG) sensory axons and spinal motor axons, forms the dorsal ramus projecting to the dorsal musculature. By using the free-floating immunohistochemistry method, we closely examined the spatiotemporal pattern of the formation of the dorsal ramus and the relationship between its projection to the myotome/dorsal musculature and semaphorin 3A (Sema3A), which is an axonal guidance molecule. In embryonic day (E) 10.5-E11.5 wild-type mouse embryos, we clearly showed the existence of a waiting period for the dorsal ramus projection to the myotome. In contrast, in E10.5-E11.5 Sema3A-deficient embryos, the dorsal ramus fibers projected beyond the edge of the myotome without exhibiting the waiting period for projection. These results strongly suggest that the delayed innervation by dorsal ramus fibers may be caused by Sema3A-induced axon repulsion derived from the myotome. Next, by performing culture experiments, we confirmed that E12.5 mouse axons responded to Sema3A-induced repulsion. Together, our results imply that Sema3A may play a key role in the proper development of the dorsal ramus projection.

Evolutionary divergence of trigeminal nerve somatotopy in amniotes.

The trigeminal circuit relays somatosensory input from the face into the central nervous system. In central nuclei, the spatial arrangement of neurons reproduces the physical distribution of peripheral receptors, thus generating a somatotopic facial map during development. In mice, the ophthalmic, maxillary, and mandibular trigeminal nerve branches maintain a somatotopic segregation and generate spatially organized patterns of connectivity within hindbrain target nuclei. To investigate conservation of somatotopic organization, we compared trigeminal nerve organization in turtle, chick, and mouse embryos. We found that, in the turtle, mandibular and maxillary ganglion neuron rostrocaudal segregation and trigeminal tract somatotopy are similar to mouse. In contrast, chick mandibular ganglion neurons are located rostrally to maxillary neurons, with some intermingling, supporting previous observations (Noden [1980], J Comp Neurol 190:429-444). This organization results in an inversion of the relative positions and less precise axonal sorting of the maxillary and mandibular branches within the trigeminal tract, as compared to mouse and turtle. Moreover, using the turtle and chick orthologs of Drg11 in combination with Hoxa2 expression and axonal tracings from the periphery, we mapped the chick PrV nucleus position to rhombomere 1, confirming previous studies (Marin and Puelles [1995], Eur J Neurosci 7:1714-1738) and in contrast to mouse PrV, which mainly maps to rhombomere 2-3 (Oury et al. [2006], Science 313:1408-1413). Thus, somatotopy of trigeminal ganglion and nerve organization is only partially conserved through amniote evolution, possibly in relation to the modification of facial somatosensory structures and morphologies.

Single-neuron diversity generated by Protocadherin-ß cluster in mouse central and peripheral nervous systems.

The generation of complex neural circuits depends on the correct wiring of neurons with diverse individual characteristics. To understand the complexity of the nervous system, the molecular mechanisms for specifying the identity and diversity of individual neurons must be elucidated. The clustered protocadherins (Pcdh) in mammals consist of approximately 50 Pcdh genes (Pcdh-α, Pcdh-ß, and Pcdh-γ) that encode cadherin-family cell surface adhesion proteins. Individual neurons express a random combination of Pcdh-α and Pcdh-γ, whereas the expression patterns for the Pcdh-ß genes, 22 one-exon genes in mouse, are not fully understood. Here we show that the Pcdh-ß genes are expressed in a 3'-polyadenylated form in mouse brain. In situ hybridization using a pan-Pcdh-ß probe against a conserved Pcdh-ß sequence showed widespread labeling in the brain, with prominent signals in the olfactory bulb, hippocampus, and cerebellum. In situ hybridization with specific probes for individual Pcdh-ß genes showed their expression to be scattered in Purkinje cells from P10 to P150. The scattered expression patterns were confirmed by performing a newly developed single-cell 3'-RACE analysis of Purkinje cells, which clearly demonstrated that the Pcdh-ß genes are expressed monoallelically and combinatorially in individual Purkinje cells. Scattered expression patterns of individual Pcdh-ß genes were also observed in pyramidal neurons in the hippocampus and cerebral cortex, neurons in the trigeminal and dorsal root ganglion, GABAergic interneurons, and cholinergic neurons. Our results extend previous observations of diversity at the single-neuron level generated by Pcdh expression and suggest that the Pcdh-ß cluster genes contribute to specifying the identity and diversity of individual neurons.

Effect of heavy oil on the development of the nervous system of floating and sinking teleost eggs.

Heavy oil (HO) on the sea surface penetrates into fish eggs and prevents the normal morphogenesis. To identify the toxicological effects of HO in the context of the egg types, we performed exposure experiments using floating eggs and sinking eggs. In the course of development, HO-exposed embryos of floating eggs showed abnormal morphology, whereas early larva of the sinking eggs had almost normal morphology. However, the developing peripheral nervous system of sinking eggs showed abnormal projections. These findings suggest that HO exposed fishes have problems in the developing neurons, although they have no morphological malformations. Through these observations, we conclude that HO is strongly toxic to floating eggs in the morphogenesis, and also affect the neuron development in both floating and sinking eggs.

Disruption of Sema3A expression causes abnormal neural projection in heavy oil exposed Japanese flounder larvae.

It has been well known that oil spills cause serious problems in the aquatic organisms. In particular, some species of teleosts, which develop on the sea surface thought to be affected by heavy oil (HO). During the embryogenesis, the nervous system is constructed. Therefore, it is important to study the toxicological effects of HO on the developing neurons. We exposed HO to eggs of Japanese flounder (Paralichthys olivaceus) and investigated the neural disorder. In larvae exposed by HO at the concentration of 8.75 mg/L, the facial and lateral line nerves partially entered into the incorrect region and the bundle was defasciculated. Furthermore, in the HO-exposed larvae, Sema3A, a kind of axon guidance molecule, was broadly expressed in second pharyngeal arch, a target region of facial nerve. Taken together, we suggested the possibility that the abnormal expression of Sema3A affected by HO exposure causes disruption of facial nerve scaffolding.

Nervous system disruption and concomitant behavioral abnormality in early hatched pufferfish larvae exposed to heavy oil.

BACKGROUND AND OBJECTIVE: Spills of heavy oil (HO) over the oceans have been proven to have an adverse effect on marine life. It has been hypothesized that exposure of early larvae of sinking eggs to HO leads largely to normal morphology, whereas abnormal organization of the developing neural scaffold is likely to be found. HO-induced disruption of the nervous system, which controls animal behavior, may in turn cause abnormalities in the swimming behavior of hatched larvae. To clarify the toxicological effects of HO, we performed exposure experiments and morphological and behavioral analyses in pufferfish (Takifugu rubripes) larvae. EXPERIMENTAL PROCEDURE: Fertilized eggs of pufferfish were exposed to 50 mg/L of HO for 8 days and transferred to fresh seawater before hatching. The hatched larvae were observed for their swimming behavior, morphological appearance, and construction of muscles and nervous system. RESULTS AND DISCUSSION: In HO-exposed larvae, we did not detect any anomaly of body morphology. However, they showed an abnormal swimming pattern and disorganized midbrain, a higher center controlling movement. Our results suggest that HO-exposed fishes suffer developmental disorder of the brain that triggers an abnormal swimming behavior and that HO may be selectively toxic to the brain and cause physical disability throughout the life span of these fishes.