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.

A time-course study of immune response in Japanese flounder Paralichthys olivaceus exposed to heavy oil.

PURPOSE: The immunotoxicities of oil and its components on fish immunities have been investigated, but there is little literature on the recovery of the fish from the immune suppression. Therefore, the recovery of Japanese flounder Paralichthys olivaceus from an immunosuppressive effect due to heavy oil (HO) exposure was investigated in this study. METHODS: Fish were exposed to HO at a concentration of 0.385 g/L for 2 days, while control fish received no exposure. Seven fish were sampled at 0, 3, 7, and 14 days post-exposure. The respiratory rate was measured everyday as an indicator of the acute effect of HO exposure. Fish serum was collected and used for antibacterial activity assay against Edwardsiella tarda. Expression changes of respiratory and immune-related genes were evaluated by real-time PCR. RESULTS AND DISCUSSION: The respiratory rate was significantly increased in the HO-exposed group until 4 days post-exposure. A respiratory-related gene, ß-hemoglobin, was also significantly downregulated in the spleen both at 0 and 7 days post-exposure and kidney at 3 days post-exposure in HO-exposed fish. Immunotoxicity, including suppression of antibacterial activities and downregulation of the IgM gene, was observed in HO-exposed fish until 3 days post-exposure, but not after that time. From these results, we conclude that the fish likely return to normal status around 1 week.

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.

Heavy oil exposure induces high moralities in virus carrier Japanese flounder Paralichthys olivaceus.

The relationship between chemical exposure and disease outbreak in fish has not been fully defined due to the limitations of experimental systems (model fish and pathogens). Therefore, we constructed a system using the Japanese flounder, Paralichthys olivaceus, and viral haemorrhagic septicemia virus (VHSV), and evaluated it by heavy oil (HO) exposure. The fish were exposed to HO at 0.3, 0.03, 0.003, and 0 g/L following VHSV infection at doses of 10(2.5) or 10(3.5) tissue culture infectious dose (TCID)50/fish. As a result, groups given the dual stressors showed more than 90% mortality. Although VHSV infection at 10(2.5) and 10(3.5) TCID50/fish without HO exposure also induced high mortality, at 68.8% and 81.3%, respectively, HO exposure induced faster and higher mortality in the virus carrier fish, indicating that chemical stressors raise the risk of disease outbreak in fish. The experimental system established in this study could be useful for chemical risk assessment.

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.

Toxicogenomic analysis of immune system-related genes in Japanese flounder (Paralichthys olivaceus) exposed to heavy oil.

Heavy oil contamination is one of the most important environmental issues. Toxicities of polycyclic aromatic hydrocarbons (PAHs), including immune toxicities, are well characterized, however, the immune toxic effects of heavy oil, as a complex mixture of PAHs, have not been investigated. In the present study, we selected Japanese flounder (Paralichthys olivaceus) as a model organism, and observed alteration of immune function by the exposure to heavy oil. To analyze the expression profiles of immune system-related genes, we selected 309 cDNAs from our flounder EST library, and spotted them on a glass slide. Using this cDNA array, alteration of gene expression profiles was analyzed in the kidneys of flounders exposed to heavy oil. Six Japanese flounders (mean body weight: 197 g) were acclimated to laboratory conditions at 19-20 degrees C. Three fish were exposed to heavy oil C (bunker C) at a concentration of 3.8 g/L for 3 days, and the others were kept in seawater without heavy oil and used as the control. After the exposure period, the fish were transferred into control seawater and maintained for 4 days, and then they were dissected and their kidneys were removed. Total RNA was extracted from the kidney samples to use in gene expression analyses. The microarray detected alteration of immune system-related genes in the kidneys of heavy oil-exposed flounders, including down-regulation of immunoglobulin light chain, CD45, major histocompatibility complex class II antigens and macrophage colony-stimulating factor precursor, and up-regulation of interleukin-8 and lysozyme. These results suggest that pathogen resistance may be weakened in heavy oil-exposed fish, causing a subsequent bacterial infection, and then proinflammatory genes may be induced as a defensive response against the infection. Additionally, we found candidate genes for use as biomarkers of heavy oil exposure, such as N-myc downstream regulated gene 1 and heat shock cognate 71 kDa proteins.

Effects of heavy oil in the developing spotted halibut, Verasper variegatus.

It is well known that heavy oil (HO) on the sea surface causes serious problems in the aquatic environment. In particular, some species of teleosts which develop on the sea surface are thought to be affected by the HO which flows out from tankers or coastal industry. However, the toxicological effects of HO are not fully understood. We performed exposure experiments using the Pleuronectiformean fish, spotted halibut (Verasper variegatus), which is an important fishery resource in Japan. In course of the development, HO-exposed embryos showed remarkable delay in developmental processes including somite formation. We further observed abnormal development of the head morphology. Notably, treated embryos had relatively small eyes and craniofacial structures. These findings strongly suggest that HO seriously affects the cell proliferation and differentiation of the embryo. In addition, HO-exposed embryos showed abnormal neuronal development. We also performed the exposure in the larval stage. Treatment of post-hatching larvae with HO resulted in significantly greater mortality compared with controls. Through these observations, we finally conclude that HO is strongly toxic to halibut in their early life stages.

Does heavy oil pollution induce bacterial diseases in Japanese flounder Paralichthys olivaceus?

As basic research for the effect of heavy oil on the fish immune system, in this study, the number of leukocyte was counted in Japanese flounder Paralichthys olivaceus, after exposure to heavy oil at a concentration of 30 g/8L for 3 days. To compare the numbers of bacteria in the skin mucus between oil-exposed and control fish, viable bacteria were enumerated by counting colony forming unit (CFU). Compared with 5.79+/-1.88 x 10(7)leukocytes/mL in the controls, the exposed fish demonstrated higher counts, averaging 1.45+/-0.45 x 10(8)cells/mL. The bacterial numbers of control fish were 4.27+/-3.68 x 10(4)CFU/g, whereas they were 4.58+/-1.63 x 10(5)CFU/g in the exposed fish. The results suggest that immune suppression of the fish occurred due to heavy oil stressor, and bacteria could invade in the mucus, resulting in the increasing leukocyte number to prevent infectious disease.

Hoxa2- and rhombomere-dependent development of the mouse facial somatosensory map.

In the mouse trigeminal pathway, sensory inputs from distinct facial structures, such as whiskers or lower jaw and lip, are topographically mapped onto the somatosensory cortex through relay stations in the thalamus and hindbrain. In the developing hindbrain, the mechanisms generating such maps remain elusive. We found that in the principal sensory nucleus, the whisker-related map is contributed by rhombomere 3-derived neurons, whereas the rhombomere 2-derived progeny supply the lower jaw and lip representation. Moreover, early Hoxa2 expression in neuroepithelium prevents the trigeminal nerve from ectopically projecting to the cerebellum, whereas late expression in the principal sensory nucleus promotes selective arborization of whisker-related afferents and topographic connectivity to the thalamus. Hoxa2 inactivation further results in the absence of whisker-related maps in the postnatal brain. Thus, Hoxa2- and rhombomere 3-dependent cues determine the whisker area map and are required for the assembly of the whisker-to-barrel somatosensory circuit.

Segmental development of reticulospinal and branchiomotor neurons in lamprey: insights into the evolution of the vertebrate hindbrain.

During development, the vertebrate hindbrain is subdivided along its anteroposterior axis into a series of segmental bulges called rhombomeres. These segments in turn generate a repeated pattern of rhombomere-specific neurons, including reticular and branchiomotor neurons. In amphioxus (Cephalochordata), the sister group of the vertebrates, a bona fide segmented hindbrain is lacking, although the embryonic brain vesicle shows molecular anteroposterior regionalization. Therefore, evaluation of the segmental patterning of the central nervous system of agnathan embryos is relevant to our understanding of the origin of the developmental plan of the vertebrate hindbrain. To investigate the neuronal organization of the hindbrain of the Japanese lamprey, Lethenteron japonicum, we retrogradely labeled the reticulospinal and branchial motoneurons. By combining this analysis with a study of the expression patterns of genes identifying specific rhombomeric territories such as LjKrox20, LjPax6, LjEphC and LjHox3, we found that the reticular neurons in the lamprey hindbrain, including isthmic, bulbar and Mauthner cells, develop in conserved rhombomere-specific positions, similar to those in the zebrafish. By contrast, lamprey trigeminal and facial motor nuclei are not in register with rhombomere boundaries, unlike those of gnathostomes. The trigeminal-facial boundary corresponds to the rostral border of LjHox3 expression in the middle of rhombomere 4. Exogenous application of retinoic acid (RA) induced a rostral shift of both the LjHox3 expression domain and branchiomotor nuclei with no obvious repatterning of rhombomeric segmentation and reticular neurons. Therefore, whereas subtype variations of motoneuron identity along the anteroposterior axis may rely on Hox-dependent positional values, as in gnathostomes, such variations in the lamprey are not constrained by hindbrain segmentation. We hypothesize that the registering of hindbrain segmentation and neuronal patterning may have been acquired through successive and independent stepwise patterning changes during evolution.