Disruption of circulation by ethanol promotes fetal alcohol spectrum disorder (FASD) in medaka (Oryzias latipes) embryogenesis

Japanese medaka (Oryzias latipes) embryos exposed to ethanol have developed craniofacial, cardiovascular and skeletal defects which can be compared with the phenotypic features of fetal alcohol spectrum disorder (FASD) observed in human. The present experiment was designed to show that the disruption in circulation by ethanol during embryogenesis is a potential cause of FASD. Fertilized eggs were exposed to ethanol (0, 100 and/or 400 mM) for 24 or 48 h at various developmental stages (Iwamatsu stages 4-30) and were analyzed at 6 day post fertilization (dpf). It was observed that controls and the embryos exposed to 100 mM ethanol were in circulating state; however, a significant number of embryos of stages 4-24 exposed to 400 mM ethanol had disrupted circulation. Compared to controls, protein and RNA contents were significantly reduced in non-circulating embryos. Lipid peroxidation (LPO) analysis was made at 3, 6, 24, 48, 96 and 144 hour post fertilization (hpf). LPO was increased with the advancement of morphogenesis; however, ethanol or the circulation status had no effect. We further analyzed alcohol dehydrogenase (Adh 5 and adh8) and aldehyde dehydrogenase (Aldh9A and Aldh1A2) enzyme mRNAs in the embryos exposed to 400 mM ethanol for 24 h. A developmental stage-specific reduction in these enzyme mRNAs by ethanol was observed. We conclude that ethanol-induced disruption in circulation during embryogenesis is a potential cause of the development of FASD features in medaka.     

Valproate-induced teratogenesis in Japanese rice fish (Oryzias latipes) embryogenesis

Fertilized eggs of Japanese rice fish (medaka) at three developmental stages (Iwamatsu stages 4-30) were exposed to waterborne valproic acid (VPA) (0-80 mM) in hatching solution for 48 h. The amount of valproate to cause 50% mortality (IC(50)) is found to be developmental stage-specific. The embryos were more sensitive to valproate at early stages of development (Iwamatsu stages 4-10) than in the embryos in late stages (Iwamatsu stages 17-30). Valproate exposed embryos have microcephaly and disrupted cardiovasculature with delayed vessel circulation, thrombus formation, and slow heart rate. The hatching efficiency is also reduced by valproate exposure due to developmental delay. The mRNA analysis of nine genes belong to oxidative stress (catalase, gsr, gst), neurogenesis (iro3, wnt1, shh, otx2, nlgn3b) and cell cycle regulation (ccna2) have been done. It was observed that the genes belong to oxidative stress remained unaltered after valproate exposure. However, some of the genes belong to neurogenesis (wnt1,shh, otx2 and nlgn3b) and cell cycle (ccna2) showed developmental stage-specific alteration after valproate exposure. This study indicates that valproate is able to induce some of the phenotypic features which are analogous to human fetal valproate syndrome (FVS). Modulation of genes expressed in neural tissues indicates that this fish can be used to analyze the mechanisms of many neurobehavioral disorders like Autism spectrum disorder (ASD) in human.     

Modulation of ethanol toxicity by Asian ginseng (Panax ginseng) in Japanese ricefish (Oryzias latipes) embryogenesis

Alcohol consumption by women during pregnancy often induces fetal alcohol spectrum disorder (FASD) in children who have serious central nervous system (CNS), cardiovascular, and craniofacial defects. Prevention of FASD, other than women abstaining from alcohol drinking during pregnancy, is not known. A limitation of the use of synthetic anti-alcoholic drugs during pregnancy led us to investigate herbal products. In particular, many plants including Asian ginseng (Panax ginseng) have therapeutic potential for the treatment of alcoholism. We used Japanese ricefish (medaka) (Oryzias latipes), an animal model of FASD, for identifying herbal medicines that can attenuate ethanol toxicity. Fertilized eggs in standard laboratory conditions were exposed to ginseng (PG) root extract (0–2 mg/mL) either 0–2 (group A) or 1–3 (group B) day post fertilization (dpf) followed by maintenance in a clean hatching solution. The calculated IC₅₀ as determined 10 dpf in A and B groups were 355.3 ± 1.12 and 679.7 ± 1.6 μg/mL, respectively. Simultaneous exposure of embryos in sub-lethal concentrations of PG (50–200 μg/mL) and ethanol (300 mM) for 48 h disrupted vessel circulation and enhanced mortality. However, PG (100 μg/mL) may partially protect trabecular cartilage (TC) deformities in the neurocranium in B group embryos induced by ethanol (300 mM). To understand the mechanism, embryonic ethanol concentration was measured at 2 dpf and adh5, adh8, aldh2, aldh9a, catalase, GST, and GR mRNAs were analyzed at 6 dpf. It was observed that although ethanol is able to reduce adh8 and GST mRNA contents, the simultaneous addition of PG was unable to alter ethanol level as well as mRNA contents in these embryos. Therefore, antagonistic effects of PG on ethanol toxicity are mediated by a mechanism which is different from those regulating ethanol metabolism and oxidative stress.     

Japanese medaka (Oryzias latipes): developmental model for the study of alcohol teratology

BACKGROUND: Animal models are necessary to investigate the mechanism of alcohol-induced birth defects. We have used Japanese medaka (Oryzias latipes) as a non-mammalian model to elucidate the molecular mechanism(s) of ethanol teratogenesis. METHODS: Medaka eggs, within 1 hr post-fertilization (hpf) were exposed to waterborne ethanol (0-1000 mM) in hatching solution for 48 hr. Embryo development was observed daily until 10 days post-fertilization (dpf). The concentration of embryonic ethanol was determined enzymatically. Cartilage and bones were stained by Alcian blue and calcein, respectively and skeletal and cardiovascular defects were assessed microscopically. Genetic gender of the embryos was determined by PCR. Levels of two isoenzymes of alcohol dehydrogenase (Adh) mRNAs were determined by semi-quantitative and real-time RT-PCR. RESULTS: The concentration of ethanol required to cause 50% mortality (LC50) in 10 dpf embryos was 568 mM, however, the embryo absorbed only 15-20% of the waterborne ethanol at all ethanol concentrations. The length of the lower jaw and calcification in tail fin cartilaginous structures were reduced by ethanol exposure. Active blood circulation was exhibited at 50+ hpf in embryos treated with 0-100 mM ethanol; active circulation was delayed and blood clots developed in embryos treated with 200-400 mM ethanol. The deleterious effects of ethanol were not gender-specific. Moreover, ethanol treatment was unable to alter the constitutive expression of either Adh5 or Adh8 mRNA in the medaka embryo. CONCLUSIONS: Preliminary results suggested that embryogenesis in medaka was significantly affected by ethanol exposure. Phenotypic features normally associated with ethanol exposure were similar to that observed in mammalian models of fetal alcohol syndrome. The results further indicated that medaka embryogenesis might be used as an alternative non-mammalian model for investigating specific alterations in gene expression as a means to understand the molecular mechanism(s) of ethanol-induced birth defects.     

Zebrafish zic1 expression in brain and somites is affected by BMP and hedgehog signalling.

Here we report the expression of the zebrafish zic1 gene, also known as opl, a homologue to other vertebrate Zic genes and the Drosophila odd-paired gene. zic1 expression starts during epiboly stages in lateral parts of the neural plate and eventually comes to lie in dorsal regions of the developing brain following the morphogenetic movements of neural tube formation. To address the question whether BMP2 signalling affects the extent of zic1 expression, we analysed swirl and chordino mutant embryos. Expanded Zic1 expression in swirl and reduced expression in chordino as well as in bmp2 injected embryos suggest that BMP2 and its antagonists define the extent of zic1 expression in the neural plate. By searching for factors responsible for the dorsal restriction of Zic1 expression, we found zic1 expression is eliminated in sonic hedgehog (shh) injected embryos. The most rostral expression however is not affected by Shh suggesting that Shh plays a different role in dorso-ventral patterning of the future telencephalon. During somitogenesis zic1 is expressed in the dorsal most part of the developing somites. Here zic1 marks cells that are distinct from the main adaxial somite portion, the future myomere. zic1 expression in the somites is expanded in swirl but reduced in shh injected embryos, suggesting these factors have opposing activity in dorsoventral patterning of the somites. Later, a growing mass of zic1 expressing cells occurs in a dorsal mesenchyme that eventually invades the dorsal fin fold, suggesting a somitic contribution to the dorsal fin mesenchyme.     

Ethanol-induced attenuation of oxidative stress is unable to alter mRNA expression pattern of catalase, glutathione reductase, glutathione-S-transferase (GST1A), and superoxide dismutase (SOD3) enzymes in Japanese rice fish (Oryzias latipes) embryogenesis

Although the mechanism of ethanol toxicity during embryogenesis is unknown, our earlier studies on Japanese rice fish (Oryzias latipes) embryos indicated that the effects might be mediated through oxidative stress. In this study we have determined the oxidative stress and the mRNA content of four antioxidant enzymes (catalase, glutathione reductase, glutathione-S-transferase, and superoxide dismutase) during Japanese rice fish embryogenesis (from 0 day post-fertilization to hatching) and after exposing the embryos to ethanol (100 and 300 mM) for 48 h at three stages (0-2, 1-3 and 4-6 days post-fertilization, dpf) of organogenesis. We observed that oxidative stress was minimal in blastula, gastrula or neurula stages, increased gradually with the advancement of morphogenesis and reached its maximum level in hatchlings. The antioxidant enzyme mRNAs were constitutively expressed throughout development; however, the expression pattern was not identical among the enzymes. Catalase and superoxide dismutase (SOD) mRNAs were minimal in the fertilized eggs, but increased significantly in 1 dpf and then either sharply dropped (SOD) or maintained a steady-state (catalase). Glutathione-S-transferase (GST) was very high in fertilized eggs and sharply dropped 1 dpf and then gradually increased thereafter. Glutathione reductase (GR) maintained a steady-state throughout the development. Ethanol was able to attenuate oxidative stress in embryos exposed only to 300 mM 1-3 dpf; no significant difference with controls was observed in other ethanol-treated groups. The antioxidant enzyme mRNAs also remained unaltered after ethanol treatment. From these data we conclude that the attenuation of oxidative stress by ethanol is probably due to the inhibition of normal growth of the embryos rather than by inhibiting catalase, GST, GR or SOD-dependent activities.     

Comparative analysis of a 229-kb medaka genomic region, containing the zic1 and zic4 genes, with Fugu, human, and mouse

Medaka is one of the prominent model animals, which also include other fishes such as Fugu and zebrafish. Its genome is relatively compact but has not been well characterized. Here we have sequenced a 229-kb region of medaka, containing the Double anal fin (Da) locus, and compared its structure to those in Fugu, human, and mouse. This region, representing a gene-poor region, contains no major rearrangements and can be readily compared among different species. Comparison of G+C contents and repeats suggested that medaka and Fugu are highly related as expected and that medaka is more similar to mammals than Fugu is. Sequence comparisons of developmental genes zic1 and zic4, identified within this region, revealed that zic1, but not zic4, is highly conserved among vertebrates. The 5' coding region of zic4 is, however, extremely homologous among fishes with little synonymous substitutions, implying its distinct function in fish.     

The zic1 gene is an activator of Wnt signaling

The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, we asked whether Xenopus Zic1 acted through the Wnt pathway. Using Wnt signaling inhibitors, we demonstrate that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated here. Together, our data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates.     

Hremx, the ascidian homologue of ems/emx, is expressed in the anterior and posterior-lateral epidermis but not in the central nervous system during embryogenesis

Recent comparative studies on expression patterns of homeobox genes in the development between ascidians and vertebrates have come to suggest a possibility that a common basic mechanism may exist in the patterning of the central nervous system (CNS). The ems/emx genes have been demonstrated to be involved in the formation and patterning of the anterior CNS in Drosophila and vertebrate embryos. In the present study, we have isolated and analyzed expression of Hremx, the ascidian homologue of ems/emx with particular attention to whether it is expressed in the larval ascidian CNS. Expression of Hremx was detected in the anterior trunk and lateral tail epidermis but not in the anterior CNS. The two expression domains of the epidermis responded in different ways upon treatment with retinoic acid: the anterior expression domain was unaltered, while the posterior expression domain extended to the anterior. The present result suggests that Hremx may have a function in anterior patterning but not in the patterning of the CNS in the ascidian embryo. We suggest the possibility that the function of ems/emx genes in the patterning of the anterior CNS in Drosophila and vertebrate embryos might have been acquired independently in the lineages to Drosophila and vertebrates.     

Development of the endoderm and gut in medaka, Oryzias latipes

We performed an extensive analysis of endodermal development and gut tube morphogenesis in the medaka embryo by histology and in situ hybridization. The markers used in these analyses included sox17, sox32, foxA2, gata-4, -5, -6 and shh. sox17, sox32, foxA2, and gata-5 and -6 are expressed in the early endoderm to the onset of gut tube formation. Sections of medaka embryos hybridized with foxA2, a pan-endodermal marker during gut morphogenesis, demonstrated that gut tube formation is initiated in the anterior portion and that the anterior and mid/posterior gut undergo distinct morphogenetic processes. Tube formation in the anterior endoderm that is fated to the pharynx and esophagus is much delayed and appears to be independent of gut morphogenesis. The overall aspects of medaka gut development are similar to those of zebrafish, except that zebrafish tube formation initiates at both the anterior and posterior portions. Our results therefore describe both molecular and morphological aspects of medaka digestive system development that will be necessary for the characterization of medaka mutants.     

Controlled Cre/loxP Site-Specific Recombination in the Developing Brain in Medaka Fish,Oryzias latipes

Background

Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.

Methodology/Principal Findings

To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0–1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2–3 dpf embyos compared with 0–1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.

Conclusions/Significance

We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.     

Ontogeny of the circadian system during embryogenesis in rainbow trout (Oncorhynchus mykyss) and the effect of prolonged exposure to continuous illumination on daily rhythms of per1, clock, and aanat2 expression

It is widely held that the development of the circadian system during embryogenesis is important for future survival of an organism. Work in teleosts has been, to date, limited to zebrafish, which provides little insight into the diversity of this system within such a large vertebrate class. In this study, the authors analyzed the diel expression of per1, clock, and aanat2 in unfertilized rainbow trout oocytes and embryos maintained under either a 12:12-h light:dark (LD) cycle or continuous illumination (LL) from fertilization. 24-h profiles in expression were measured at fertilization as well as 8, 21 42, and 57 days postfertilization (dpf). Both per1 and clock were expressed in unfertilized oocytes and all embryonic stages, whereas aanat2 expression was only measureable from 8 dpf. A reduction in both per1 and clock mean expression levels between unfertilized oocytes/0-1 dpf embryos and 8-9 dpf embryos was suggestive of a transition from maternal RNA to endogenous mRNA expression. Although aanat2 expression was not clearly associated with photic conditions, photoperiod treatment did alter the expression of per1 and clock expression/rhythmicity from as early as 8 dpf (per1), which could suggest the presence and functionality of an as yet unidentified "photoreceptor." As a whole, this work demonstrates that clock systems are present and functional during embryonic development in rainbow trout. Further studies of their expression and regulation will help understand how the environment interacts with embryonic development in the species.     

Expressions of Raldh3 and Raldh4 during zebrafish early development

Retinoic acid (RA) plays crucial roles in vertebrate embryogenesis. Four retinal dehydrogenases (Raldhs) that are responsible for RA synthesis have been characterized in mammals. However, only Raldh2 ortholog is identified in zebrafish. Here, we report the identification of raldh3 and raldh4 genes in zebrafish. The predicted proteins encoded by zebrafish raldh3 and raldh4 exhibit 70.0% and 73.5% amino acid identities with mouse Raldh3 and Raldh4, respectively. RT-PCR analyses reveal that both raldh3 and raldh4 mRNAs are present in early development. However, whole mount in situ hybridization shows that raldh3 mRNA is first expressed in the developing eye region of zebrafish embryos at 10-somite stage. At 24 hpf (hours post fertilization), raldh3 mRNA is expressed in the ventral retina of eye. At 36 hpf, the mRNA is also expressed in otic vesicle in addition to ventral retina, and it maintains its expression pattern till 2 dpf (days post fertilization). At 3 dpf, raldh3 mRNA becomes very weak in ventral retina but is present in otic vesicle at a high level. At 5 dpf and 7 dpf, raldh3 is no longer expressed in eyes but is expressed in otic vesicles at a very low level. raldh4 mRNA is initially detected in developing liver and intestine regions at 2 dpf embryos. Its expression level becomes very high in these two regions of embryos from 3 dpf to 5 dpf. Analysis on the sections of 5 dpf embryos reveals that raldh4 is expressed in the epithelium of intestine. At 7 dpf, raldh4 mRNA is only weakly expressed in the epithelium of intestinal bulb.     

Disrupting the establishment of polarizing activity by teratogen exposure.

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.