Association between periodontal inflammation and serum lipid profile in a healthy population: A cross-sectional study.

AIMS: The impact of periodontal inflammation on lipid metabolism is controversial. This study aimed to investigate the association between full-mouth periodontal inflammation and serum lipid levels. MATERIALS AND METHODS: In this cross-sectional study, we performed periodontal and bacteriological examinations during medical checkup on 131 subjects. The association between the periodontal inflamed surface area (PISA) and the lipid markers was analyzed by multiple linear regression, adjusting for age, sex, smoking, and body mass index. RESULTS: Overall, 118 medically healthy participants were analyzed. The proportions of none, mild, moderate, and severe periodontitis were 37.3%, 32.2%, 25.4%, and 5.1%, respectively. Multivariate analysis showed that high-density lipoprotein cholesterol was significantly higher in participants with the lowest tertile of PISA values (PISA low, coefficient: 7.94; 95% confidence interval [CI]: 1.63, 14.26, p = .01) compared to those in other tertiles (PISA high). Low-density/high-density lipoprotein cholesterol and total/high-density lipoprotein cholesterol ratios were significantly lower in the PISA-low group than the PISA-high group (coefficient: -0.26 and -0.30; 95% CI: -0.50, -0.02, and -0.59, -0.0002; p = .04 and .0498). Serum high-sensitivity C-reactive protein level, but not serum Porphyromonas gingivalis antibody titer, partly explained the association between PISA and high-density lipoprotein cholesterol. A significant interaction between female sex and PISA values toward high-density lipoprotein cholesterol level was detected. CONCLUSION: Periodontal inflammation was inversely associated with higher high-density lipoprotein cholesterol, especially in females. Elevated serum C-reactive protein partly explained this association.

Abscopal Activation of Microglia in Embryonic Fish Brain Following Targeted Irradiation with Heavy-Ion Microbeam.

Microglia remove apoptotic cells by phagocytosis when the central nervous system is injured in vertebrates. Ionizing irradiation (IR) induces apoptosis and microglial activation in embryonic midbrain of medaka (Oryzias latipes), where apolipoprotein E (ApoE) is upregulated in the later phase of activation of microglia In this study, we found that another microglial marker, l-plastin (lymphocyte cytosolic protein 1), was upregulated at the initial phase of the IR-induced phagocytosis when activated microglia changed their morphology and increased motility to migrate. We further conducted targeted irradiation to the embryonic midbrain using a collimated microbeam of carbon ions (250 µm diameter) and found that the l-plastin upregulation was induced only in the microglia located in the irradiated area. Then, the activated microglia might migrate outside of the irradiated area and spread through over the embryonic brain, expressing ApoE and with activated morphology, for longer than 3 days after the irradiation. These findings suggest that l-plastin and ApoE can be the biomarkers of the activated microglia in the initial and later phase, respectively, in the medaka embryonic brain and that the abscopal and persisted activation of microglia by IR irradiation could be a cause of the abscopal and/or adverse effects following irradiation.

Regular heartbeat rhythm at the heartbeat initiation stage is essential for normal cardiogenesis at low temperature.

BACKGROUND: The development of blood flow in the heart is crucial for heart function and embryonic survival. Recent studies have revealed the importance of the extracellular matrix and the mechanical stress applied to the valve cushion that controls blood flow to the formation of the cardiac valve during embryogenesis. However, the events that trigger such valve formation and mechanical stress, and their temperature dependence have not been explained completely. Medaka (Oryzias latipes) inhabits a wide range of East Asia and adapts to a wide range of climates. We used medaka embryos from different genomic backgrounds and analyzed heartbeat characteristics including back-and-forth blood flow and bradyarrhythmia in embryos incubated at low temperature. We also used high-speed imaging analysis to examine the heartbeat of these animals after transient exposure to low temperature. RESULTS: Embryos of the Hd-rR medaka strain exhibited back-and-forth blood flow in the heart (blood regurgitation) after incubation at 15 °C. This regurgitation was induced by exposure to low temperature around the heartbeat initiation period and was related to abnormalities in the maintenance or pattern of contraction of the atrium or the atrioventricular canal. The Odate strain from the northern Japanese group exhibited normal blood flow after incubation at 15 °C. High-speed time-lapse analysis of the heartbeat revealed that bradyarrhythmia occurred only in Hd-rR embryos incubated at 15 °C. The coefficient of contraction, defined as the quotient of the length of the atrium at systole divided by its length at diastole, was not affected in either strain. The average heart rate after removing the effect of arrhythmia did not differ significantly between the two strains, suggesting that the mechanical stress of individual myocardial contractions and the total mechanical stress could be equivalent, regardless of the presence of arrhythmia or the heart rate. Test-cross experiments suggested that this circulation phenotype was caused by a single major genomic locus. CONCLUSIONS: These results suggest that cardiogenesis at low temperature requires a constant heartbeat. Abnormal contraction rhythms at the stage of heartbeat initiation may cause regurgitation at later stages. From the evolutionary viewpoint, strains that exhibit normal cardiogenesis during development at low temperature inhabit northern environments.

Retinal regeneration after injury induced by gamma-ray irradiation during early embryogenesis in medaka, Oryzias latipes.

PURPOSE: Zebrafish, a small fish model, exhibits a multipotent ability for retinal regeneration after damage throughout its lifetime. Compared with zebrafish, birds and mammals exhibit such a regenerative capacity only during the embryonic period, and this capacity decreases with age. In medaka, another small fish model that has also been used extensively in biological research, the retina's inner nuclear layer (INL) failed to regenerate after injury in the hatchling at eight days postfertilization (dpf). We characterized the regenerative process of the embryonic retina when the retinal injury occurred during the early embryonic period in medaka. METHODS: We employed a 10 Gy dose of gamma-ray irradiation to initiate retinal injury in medaka embryos at 3 dpf and performed histopathological analyses up to 21 dpf. RESULTS: One day after irradiation, numerous apoptotic neurons were observed in the INL; however, these neurons were rarely observed in the ciliary marginal zone and the photoreceptor layer. Numerous pyknotic cells were clustered in the irradiated retina until two days after irradiation. These disappeared four days after irradiation, but the abnormal bridging structures between the INL and ganglion cell layer (GCL) were present until 11 days after irradiation, and the neural layers were completely regenerated 18 days after irradiation. After gamma-ray irradiation, the spindle-like Müller glial cells in the INL became rounder but did not lose their ability to express SOX2. CONCLUSIONS: Irradiated retina at 3 dpf of medaka embryos could be completely regenerated at 18 days after irradiation (21 dpf), although the abnormal layer structures bridging the INL and GCL were transiently formed in the retinas of all the irradiated embryos. Four days after irradiation, embryonic medaka Müller glia were reduced in number but maintained SOX2 expression as in nonirradiated embryos. This finding contrasts with previous reports that 8 dpf medaka larvae could not fully regenerate damaged retinas because of loss of SOX2 expression.

Generation of monoclonal antibodies against the Galß1-4Gal epitope: a key tool in studies of species-specific glycans expressed in fish, amphibians and birds.

Whereas the Galß1-4Gal epitope is rarely found in mammalian glycans, it has been found in glycans of various species of non-mammalian vertebrates, such as fish, amphibians and birds. Although glycans containing Galß1-4Gal in these vertebrates were detected by precise structural analysis of the glycans using mass spectrometry and/or NMR spectrometry, there are no convenient methods to detect Galß1-4Gal from various samples. To analyze systematically the distribution of Galß1-4Gal in nature, we generated mouse monoclonal antibodies (mAbs) specific for Galß1-4Gal using extracts of medaka eggs as an immunogen. Four mAbs (two immunoglobulin (Ig)Ms and two IgG1s) were obtained by enzyme-linked immunosorbent assay-based screening. The specificities of these mAbs were evaluated by frontal affinity chromatography using 142 kinds of 2-aminopyridine (PA)-derivatized oligosaccharides. While all mAbs interacted with (Galß1-4Gal)-containing oligosaccharides at their non-reducing termini with dissociation constants (K(d)) ranging from 1.0 x 10⁻5 to 2.8 x 10⁻4 M, no apparent interaction was observed with any other glycans. The number of branches containing Galß1-4Gal on N-glycans did not significantly affect K(d) of mAbs of IgG1 subclasses, but those of IgM mAbs were decreased by ∼1 order of magnitude, in increments of the number of branches present. Using the mAbs, we established that Galß1-4Gal is also expressed on glycoproteins in various tissues from the African clawed frog. Immunohistochemical staining of medaka sections revealed that Galß1-4Gal epitopes were expressed in the endothelium, epithelium and epidermis, which directly contact the external environment or invading organisms. Thus, these mAbs are useful for systematically investigating the species-specific expression of glycans, which may act as a barrier against infection.

Postpartum smoking relapse among women who quit during pregnancy: cross-sectional study in Japan.

AIM: To determine the postpartum smoking relapse rate among women in Japan who quit smoking during pregnancy and to clarify factors related to smoking relapse. METHODS: A self-administered questionnaire survey was conducted as a cross-sectional study of all mothers of children who underwent health checkups after birth in randomly selected municipalities in Japan from May to July 2009. Using valid data of 20,601 mothers, smoking rate was calculated. In addition, χ(2) -test and multiple logistic regression analysis were used to clarify related factors to the smoking relapse. RESULTS: The smoking rates among women were 15.8% at the time when they became pregnant, 5.1% during pregnancy and 11.3% after giving birth. Among women who smoked at the time they became pregnant, the smoking rate during pregnancy was 31.1%. Among women who quit smoking during pregnancy, the postpartum smoking relapse rate was 41.0%. The odds ratios (95% confidence interval) for factors in smoking relapse were 0.72 (0.60-0.88) for women spending time with their child in a relaxed mood, 0.67 (0.47-0.94) for women having someone to talk to on the Internet about childrearing, 1.94 (1.60-2.35) for women who worked and 3.37 (2.61-4.35) for women whose partner smoked after they gave birth. CONCLUSION: It is hoped that future research will establish methods to further support smoking cessation and the continuation of smoking cessation after childbirth, and develop mechanisms to spread knowledge about the harm of smoking in society and encourage women not to start.

Responses of hematopoietic cells after ionizing-irradiation in anemic adult medaka (Oryzias latipes).

PURPOSE: Hematopoietic tissues of vertebrates are highly radiation sensitive and the effects of ionizing radiation on the hematopoiesis have been studied in mammals and teleosts for decades. In this study, radiation responses in the kidney, the main hematopoietic organ in teleosts, were investigated in Japanese medaka (Oryzias latipes), which has been a model animal and a large body of knowledge has been accumulated in radiation biology. METHODS: Kidney, the main hematopoietic tissue of adult medaka fish, was locally irradiated using proton and carbon ion beams irradiation system of Takasaki Ion Accelerator for Advanced Radiation Application (TIARA), QST, and the effects on peripheral blood cells and histology of the kidney were investigated. RESULTS: When only kidneys were locally irradiated with proton or carbon ion beam (15 Gy), the hematopoietic cells in the irradiated kidney and cell density in the peripheral blood decreased 7 days after the irradiation in the same manner as after the whole-body irradiation with γ-rays (15 Gy). These results demonstrate that direct irradiation of the hematopoietic cells in the kidney induced cell death and/or cell cycle arrest and stopped the supply of erythroid cells. Then, the cell density in the peripheral blood recovered to the control level within 4 days and 7 days after the γ-ray and proton beam irradiation (15 Gy), respectively, while the cell density in the peripheral blood did not recover after the carbon ion beam irradiation (15 Gy). The hematopoietic cells in the irradiated kidneys temporarily decreased and recovered to the control level within 21 days after the γ-ray or proton beam irradiation (15 Gy), while it did not recover after the carbon ion beam irradiation (15 Gy). In contrast, the recovery of the cell density in the peripheral blood delayed when anemic medaka were irradiated 1 day after the administration of phenylhydrazine. With and without γ-ray irradiation, a large number of hematopoietic cells was still proliferating in the kidney 7 days after the anemia induction. CONCLUSIONS: The results obtained strongly suggest that the hematopoietic stem cells in medaka kidney prioritize to proliferate and increase peripheral blood cells to eliminate anemia, even when they are damaged by high-dose irradiation.

The light-dependent daily cycle of ovulation in the oviparous medaka fish, Oryzias latipes (Atherinomorpha: Beloniformes: Adrianichthyidae) artificially pregnant with developing embryos.

In the oviparous medaka fish, Oryzias latipes, mature spermatozoa that were artificially introduced into the ovarian cavity retaining ovulated eggs could internally fertilize these eggs. This enabled us to examine the effect of ovarian gestation on the ovulation cycle. Most freshly ovulated eggs could be internally fertilized in the ovarian cavity. Yet eggs ovulated 24 h after single insemination remained unfertilized in the ovarian cavity. Artificially pregnant females persisted in a daily cycle of ovulation, which occurred shortly before the onset of light under the present reproductive conditions. Females continuously ovulated a certain number of eggs despite ovarian gestation, that is, the presence of embryos within the ovarian cavity. Repeated cycles of ovulation led to crowding in the ovarian cavity because the group of fertilized eggs, with their hardened egg envelope (chorion or zona radiata), plugged the genital orifice. The development of fertilized eggs was retarded and ceased around the initiation stage of blood circulation, but when they were transferred from the ovarian cavity into regular saline, they regained their ability to develop normally up to hatching. These results show that in oviparous female medaka, ovarian gestation exerted little effect on the time of ovulation and the number of ovulated eggs.

Glycemic control and number of natural teeth: analysis of cross-sectional Japanese employment-based dental insurance claims and medical check-up data.

AIM: Diabetes mellitus is a well-known risk factor for onset and progression of periodontal disease. However, the continuous relationship between glycemic control and the number of natural teeth has not been well characterized in large-scale studies. We aimed to determine whether the glycated hemoglobin A1c (HbA1c) level and fasting plasma glucose (FPG) are associated with the number of natural teeth. METHODS: A cross-sectional study: A database comprising employment-based health insurance claim and medical check-up data from 706,150 participants between April 2015 and March 2016 in Japan. The exclusion criteria included missing data regarding dental receipts, number of natural teeth, HbA1c, smoking status, and age < 20 years. Ultimately, 233,567 individuals were analyzed. The participants were allocated to five groups according to their HbA1c and three groups according to their FPG, and then the number of natural teeth were compared. RESULTS: Higher HbA1c was associated with fewer teeth in participants ≥ 30 years of age (P for trend < 0.001). Higher FPG was associated with fewer teeth between 30 and 69 years of age (P for trend < 0.001). Participants with impaired fasting glucose was already at risk for fewer teeth between 40 and 69 years of age than those with normal FPG. CONCLUSIONS: Glycemic control is strongly associated with the number of natural teeth in the real-world setting. Furthermore, there are continuous relationships of HbA1c and FPG with number of natural teeth including individual with impaired fasting glucose. These data emphasize the importance of glycemic control and appropriate oral care for the protection against tooth loss. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13340-021-00533-2.

3D reconstructed brain images reveal the possibility of the ogg1 gene to suppress the irradiation-induced apoptosis in embryonic brain in medaka (Oryzias latipes).

The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy (137Cs, 7.3 Gy/min.) at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos (n = 3) compared to wild-type embryos (n = 3) (P = 0.04), suggesting that irradiation-induced apoptosis in medaka embryonic brain can be suppressed in the presence of functional ogg1. Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.

Real-world evidence of the impact of obesity on residual teeth in the Japanese population: A cross-sectional study.

BACKGROUND: Tooth loss is associated with nutritional status and significantly affects quality of life, particularly in older individuals. To date, several studies reveal that a high BMI is associated with tooth loss. However, there is a lack of large-scale studies that examined the impact of obesity on residual teeth with respect to age and tooth positions. OBJECTIVE: We assessed the impact of obesity on the number and position of residual teeth by age groups using large scale of Japanese database. METHODS: This was a cross-sectional study of 706150 subjects that were included in the database that combined the data from health insurance claims and health check-up, those lacking information about BMI, HbA1c level, smoking status, and the number of residual teeth were excluded. Thus, a total of 233517 aged 20-74 years were included. Subjects were classified into 4 categories based on BMI, and the number of teeth was compared between age-groups. The percentage of subjects with residual teeth in each position was compared between groups with obesity (BMI ≥25.0 kg/m2) and non-obesity. Logistic regression analysis was performed to clarify whether obesity predicts having <24 teeth. RESULTS: Higher BMI was associated with fewer teeth over 40s (P for trend <0.0001 when <70s). Obesity was associated with the reduction of residual teeth in the maxillary; specifically, the molars were affected over the age 30. Smoking status further affected tooth loss at positions that were not affected by obesity alone. After adjusting for age, sex, smoking status, and HbA1c ≥6.5%, obesity remained an independent predictive factor for having <24 teeth (ORs: 1.35, 95% CIs: 1.30-1.40). CONCLUSIONS: We found that an increase in BMI was associated with a decrease in the number of residual teeth from younger ages independently of smoking status and diabetes in the large scale of Japanese database.

Strain difference in transgene-induced tumorigenesis and suppressive effect of ionizing radiation.

Transgenic expression in medaka of the Xiphophorus oncogene xmrk, under a pigment cell specific mitf promoter, induces hyperpigmentation and pigment cell tumors. In this study, we crossed the Hd-rR and HNI inbred strains because complete genome information is readily available for molecular and genetic analysis. We prepared an Hd-rR (p53+/-, p53-/-) and Hd-rR HNI hybrid (p53+/-) fish-based xmrk model system to study the progression of pigment cells from hyperpigmentation to malignant tumors on different genetic backgrounds. In all strains examined, most of the initial hyperpigmentation occurred in the posterior region. On the Hd-rR background, mitf:xmrk-induced tumorigenesis was less frequent in p53+/- fish than in p53-/- fish. The incidence of hyperpigmentation was more frequent in Hd-rR/HNI hybrids than in Hd-rR homozygotes; however, the frequency of malignant tumors was low, which suggested the presence of a tumor suppressor in HNI genetic background fish. The effects on tumorigenesis in xmrk-transgenic immature medaka of a single 1.3 Gy irradiation was assessed by quantifying tumor progression over 4 consecutive months. The results demonstrate that irradiation has a different level of suppressive effect on the frequency of hyperpigmentation in purebred Hd-rR compared with hybrids.

Morphogenesis of the medaka cerebellum, with special reference to the mesencephalic sheet, a structure homologous to the rostrolateral part of mammalian anterior medullary velum.

We have examined cerebellar morphogenesis after neural tube stage in medaka (Oryzias latipes), a ray-finned fish, by conventional histology and immunohistochemistry using anti-proliferating cell nuclear antigen (PCNA) and anti-acetylated tubulin antibodies. Our results indicate that the medaka cerebellum is formed in 4 successive stages: (1) formation and enlargement of the cerebellar primordia; (2) rostral midline fusion of the left/right halves of the cerebellar primordia; (3) formation of the cerebellar matrix zones in the midline and caudalmost regions of the primitive cerebellum, and (4) growth and differentiation of the cerebellum. Our results also show that cerebellar morphogenesis is different from that in mammals in 3 important points: the developmental origins of the primordia, directions along which cerebellar fusion proceeds, and number, locations and duration of the cerebellar matrix zones. During the course of this study, an alar-derived membranous structure between the cerebellum and the midbrain in the adult medaka brain was identified as the structure homologous to the rostrolateral part of the mammalian anterior medullary velum. We have named this structure in the adult teleostean brains as the 'mesencephalic sheet'. The present study indicates that there exists both conserved and divergent patterns in cerebellar morphogenesis in vertebrates.

Collimated Microbeam Reveals that the Proportion of Non-Damaged Cells in Irradiated Blastoderm Determines the Success of Development in Medaka (Oryzias latipes) Embryos.

It has been widely accepted that prenatal exposure to ionizing radiation (IR) can affect embryonic and fetal development in mammals, depending on dose and gestational age of the exposure, however, the precise machinery underlying the IR-induced disturbance of embryonic development is still remained elusive. In this study, we examined the effects of gamma-ray irradiation on blastula embryos of medaka and found transient delay of brain development even when they hatched normally with low dose irradiation (2 and 5 Gy). In contrast, irradiation of higher dose of gamma-rays (10 Gy) killed the embryos with malformations before hatching. We then conducted targeted irradiation of blastoderm with a collimated carbon-ion microbeam. When a part (about 4, 10 and 25%) of blastoderm cells were injured by lethal dose (50 Gy) of carbon-ion microbeam irradiation, loss of about 10% or less of blastoderm cells induced only the transient delay of brain development and the embryos hatched normally, whereas embryos with about 25% of their blastoderm cells were irradiated stopped development at neurula stage and died. These findings strongly suggest that the developmental disturbance in the IR irradiated embryos is determined by the proportion of severely injured cells in the blastoderm.

Phenotypic analyses of a medaka mutant reveal the importance of bilaterally synchronized expression of isthmic fgf8 for bilaterally symmetric formation of the optic tectum.

Developing neural tubes are bilaterally symmetric in all vertebrate embryos, irrespective of the presence of gene networks that generate left-right asymmetry. To explore the mechanisms that underlie the bilaterally symmetric formation of the neural tube, we examined a medaka (Oryzias latipes) dominant mutant, Oot, the neural tube of which transiently lacks normal symmetry in the optic tectum. We found that spatial changes in isthmic fgf8 expression do not occur on one side of the mutant, resulting in a transient desynchronized expression that correlates with tectal asymmetry. The application of exogenous FGF8 on one side of a wild-type embryo mimics the Oot phenotype, indicating that the bilaterally equivalent expression of isthmic fgf8 is crucial for the bilaterally symmetric development of the tectum. These results suggest that tectal symmetry is not a "default" state, but rather is maintained actively by a bilaterally coupled and synchronized regulation of isthmic fgf8 expression.

Expression of brain-type fatty acid-binding protein (fabp7) in medaka during development.

Fatty acid-binding proteins (FABPs) belong to a multigene family of small intracellular proteins that bind hydrophobic ligands. Recent studies have indicated that FABP7 plays important roles in neurogenesis or neuronal migration in vertebrates. In this study, we isolated cDNA and the genomic fragment containing the fabp7 gene for medaka fish and examined the expression of the medaka fabp7 gene through the development of their central nervous system (CNS). The medaka fabp7 gene consists of four exons in approximately 1 kb of the genomic region. Its deduced amino acid sequence exhibits over 80% identity with those of other higher vertebrates. In situ hybridization analysis demonstrated that fabp7-positive cells first appear at stage 22 in a small dorsal domain of the retina, dorsal diencephalon, and rhombencephalon, then expand to the entire CNS including the retina and the spinal cord. In addition, we generated two lines of transgenic medaka with 1.7 kb upstream of the fabp7 gene combined with the enhanced-green fluorescence protein (EGFP) gene. The spatio-temporal expression patterns of EGFP in these animals were consistent with the results of in situ hybridization analysis. The result of our reporter assays with a series of truncated fabp7 promoters suggested that POU elements play a role in fabp7 expression in medaka as well as in other vertebrates. Our transgenic animal will contribute to clarifying the role of FABP7 in the development of CNS.

Early development of the cerebellum in teleost fishes: a study based on gene expression patterns and histology in the medaka embryo.

The cerebellar structures of teleosts are markedly different from those of other vertebrates. The cerebellum continues rostrally into the midbrain ventricle, forming the valvula cerebelli, only in ray-finned fishes among vertebrates. To analyze the ontogenetic processes that underlie this morphological difference, we examined the early development of the cerebellar regions, including the isthmus (mid/hindbrain boundary, MHB), of the medaka (Oryzias latipes), by histology and in-situ hybridization using two gene (wnt1 and fgf8) probes. Isthmic wnt1 was expressed stably in the caudalmost mesencephalic region in the neural tube at all developmental stages examined, defining molecularly the caudal limit of the mesencephalon. The wnt1-positive mesencephalic cells became located rostrally to the isthmic constriction at Iwamatsu's stages 25-26. Isthmic fgf8 expression changed dynamically and became restricted to the rostralmost metencephalic region at stage 24. The rostralmost part (prospective valvula cerebelli) of the fgf8-positive rostral metencephalon protruded rostrally into the midbrain ventricle, bypassing the isthmic constriction, at stages 25-26. Thus, the isthmic constriction shifted caudally with respect to the molecularly defined MHB at stages 25-26. Paired cerebellar primordia were formed from the alar plates of the fgf8-positive rostral metencephalon and the fgf8-negative caudal metencephalon in the medaka neural tube. Our results show that cerebellar development differs between teleosts and murines: both the rostral and caudal metencephalic alar plates develop into the cerebellum in medaka, whereas in the murines only the caudal metencephalic alar plate develops into the cerebellum, and the rostral plate is reduced to a thin membrane.

Rapid and simple method for quantitative evaluation of neurocytotoxic effects of radiation on developing medaka brain.

We describe a novel method for rapid and quantitative evaluation of the degree of radiation-induced apoptosis in the developing brain of medaka (Oryzias latipes). Embryos at stage 28 were irradiated with 1, 2, 3.5, and 5 Gy x-ray. Living embryos were stained with a vital dye, acridine orange (AO), for 1-2 h, and whole-mount brains were examined under an epifluorescence microscope. From 7 to 10 h after irradiation with 5 Gy x-ray, we found two morphologically different types of AO-stained structures, namely, small single nuclei and rosette-shaped nuclear clusters. Electron microscopy revealed that these two distinct types of structures were single apoptotic cells with condensed nuclei and aggregates of apoptotic cells, respectively. From 10 to 30 h after irradiation, a similar AO-staining pattern was observed. The numbers of AO-stained rosette-shaped nuclear clusters and AO-stained single nuclei increased in a dose-dependent manner in the optic tectum. We used the number of AO-stained rosette-shaped nuclear clusters/optic tectum as an index of the degree of radiation-induced brain cell death at 20-24 h after irradiation. The results showed that the number of rosette-shaped nuclear clusters/optic tectum in irradiated embryos exposed to 2 Gy or higher doses was highly significant compared to the number in nonirradiated control embryos, whereas no difference was detected at 1 Gy. Thus, the threshold dose for brain cell death in medaka embryos was taken as being between 1-2 Gy, which may not be so extraordinarily large compared to those for rodents and humans. The results show that medaka embryos are useful for quantitative evaluation of developmental neurocytotoxic effects of radiation.

Radical change of apoptotic strategy following irradiation during later period of embryogenesis in medaka (Oryzias latipes).

Induction of apoptosis in response to various genotoxic stresses could block transmission of teratogenic mutations to progeny cells. The severity of biological effects following irradiation depends on the stage at which embryos are irradiated during embryogenesis. We reported previously that irradiation of medaka embryos 3 days post fertilization (dpf) with 10 Gy of gamma rays induced high incidence of apoptotic cells in the mid-brain, however, the embryos hatched normally and developed without apparent malformations. To determine the severity of biological effects following irradiation during a later period of embryogenesis, embryos of various developmental stages were irradiated with 15 Gy of gamma rays and examined for apoptotic induction at 24 h after irradiation in the brain, eyes and pharyngeal epithelium tissues, which are actively proliferating and sensitive to irradiation. Embryos irradiated at 3 dpf exhibited many apoptotic cells in these tissues, and all of them died due to severe malformations. In contrast, embryos irradiated at 5 dpf showed no apoptotic cells and subsequently hatched without apparent malformations. Embryos irradiated at 4 dpf had relatively low numbers of apoptotic cells compared to those irradiated at 3 dpf, thereafter most of them died within 1 week of hatching. In adult medaka, apoptotic cells were not found in these tissues following irradiation, suggesting that apoptosis occurs during a restricted time period of medaka embryogenesis throughout the life. No apoptotic cells were found in irradiated intestinal tissue, which is known to be susceptible to radiation damage in mammals, whereas many apoptotic cells were found in proliferating spermatogonial cells in the mature testis following irradiation. Taken together, with the exception of testicular tissue, the results suggest a limited period during medaka embryogenesis in which irradiation-induced apoptosis can occur.

Developmental origin of diencephalic sensory relay nuclei in teleosts.

We propose here a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts based on our recent studies of gene expression patterns in the medaka (Oryzias latipes) embryonic brain and comparative hodological studies. It has been proposed that the diencephalic sensory relay system in teleosts is unique among vertebrates. Teleost relay nuclei, the preglomerular complex (PG), have been assumed to originate from the basal plate (the posterior tuberculum) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate (dorsal thalamus) of the diencephalon. Our results using in situ hybridization show, however, that many pax6- or dlx2-positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. Massive clusters of the migrated alar cells become localized in the mantle layer lateral to the posterior tubercular neuroepithelium, from which main nuclei of the PG appear to differentiate. We therefore consider most if not all neurons in the PG to be of alar, not basal, origin. Thus, the teleost PG, at least in part, can be regarded as migrated alar nuclei. Developmental and hodological data strongly suggest that the teleost PG is homologous to a part of the mammalian dorsal thalamus. The organization and origin of the diencephalic sensory relay system might have been conserved across vertebrates.