Page 1Page 2Page 3Page 4Page 51Biology 4361Developmental BiologyOctober 11, 2007Effects of ethanol on development of the Japanese MedakaBackgroundEthyl alcohol acts as a devastating teratogen in vertebrates. In the 1960s, researchers describedfetal alcohol syndrome (FAS), which is a syndrome of birth defects in the children of alcoholicmothers. Babies with FAS were characterized as having a smaller head and brain size, defects inneuronal and glial migration, extra cell death in specific brain regions, craniofacialmalformations, and heart defects. There is great variation in the ability of mothers and fetuses tometabolize ethanol, and it is thought that in some cases, even low ethanol concentrations can leadto FAS or a less severe form of FAS called fetal alcohol effect. The effects of ethanol on face andnervous system have been studied intensively in mouse and chick model systems. Mice developthe same range of developmental defects as humans when they are exposed to ethanol duringgastrulation. Typically, forebrain anomalies, including total lack of the forebrain, predominate.Many of the structures affected by FAS stem from neural crest cells, a group of cells which isderived from the dorsal neuroectoderm during embryogenesis. It has been shown that ethanolimpairs neural crest cell migration and induces their premature differentiation into facialcartilage. Ethanol can also induce apoptosis (programmed cell death) in neurons by several differentmechanisms, and it can interfere with the ability of cell adhesion molecules to function inholding cells together (Gilbert, 2006).Recently, the molecular causes of FAS have been investigated. Expression of msx2, a generequired for the development of craniofacial structures and the developing brain, was found todecrease in mice exposed to alcohol. The misexpression of the gene corresponds to FAS-inducedchanges in phenotype seen in previous research (Rifas et al., 1997). In addition, ethanol is knownto induce cyclopia in fish, and similar teratogenic effects were recently reported in othervertebrates including humans. In zebrafish, the phenotypic disturbances were found to be theresult of a loss gene expression characteristic of the ventral aspects of the fore- and midbrain(Blader and Straehle, 1988). In this experiment, we will investigate the effects of ethanol on development of the JapaneseMedaka (Oryzias latipes). Medaka embryos will be treated with varying concentrations ofethanol and phenotypes will be characterized according to gross morphology.2Procedure1. Select embryos that are at the midgastrula stage, about 50% epiboly. [Epiboly is the movementof epithelial ectoderm to enclose deeper layers of the embryo; in the medaka and otherfish, dividing animal pole cells spread over the yolk-cell in the vegetal region. The extentof movement of these cells should be apparent as you compare various embryos.]2. Prepare and label 35mm Petri dishes for control (embryo rearing medium only) and ethanol(EtOH) exposures. Add 5 ml ERM and appropriate volumes of 95% EtOH to theappropriate dishes.3. Prepare dilutions of ethanol in embryo medium. This has to be done immediately before thestart of the treatment to limit evaporation.4. Incubate embryos for 1 to 12 hours with the respective concentrations of EtOH. Longexposures should be carried out in sealed microcentrifuge tubes.5. Keep treated embryos in an incubator set to 20EC.5. Observe your embryos and record your findings over the following days (photographs,drawings). For the final evaluation of the resulting phenotype, it will be necessary todissect the embryo from the chorion. This can be accomplished by using two fine forcepsor microneedles and forcing the embryo out through a hole in the chorion.6. Fix dissected embryos for a minimum of 2 hours (or for up to two days) in Carnoy’s fixative:Glacial acetic acid 1 part100 % ethanol 3 parts7. Wash fixed embryos by replacing fixative with 70% ethanol for at least 30 minutes. Repeat70% ethanol washes a total of 3 times. Fixed embryos can be stored in 70 % ethanol in atightly sealed microcentrifuge tube.ReferencesBlader P, Straehle U: Ethanol impairs migration of the prechordal plate in the zebrafish embryo.Developmental Biology 201, 185-201 (1998)Gilbert SF: Developmental Biology, 8 ed. Sinauer Associates Inc, Sunderland, MA (2006) thRifas L, Towler DA, Avioli LV: Gestational exposure to ethanol suppresses msx2 expression indeveloping mouse embryos. Proceedings of the National Academy of Sciences USA 94, 7549-7554 (1997)1Biology 4361Developmental Biology October 11, 2007Effects of retinoic acid on development of the Japanese MedakaBackgroundRetinoic acid (RA) is a small molecule that is synthesized by most vertebrates from carotenoidpigment precursors found in various plants. Early in development, RA is important in formingthe anterior-posterior axis and in limb formation. Specifically, RA acts as a ligand for as set oftranscription factors (retinoic acid receptors; RARs) that bind to enhancers of genes regarded askey regulators of positional information during development of the primary body axis. During development, RA is secreted from a very limited group of discrete cells and affects only asmall area. Although RA is involved in normal development, it can act as a teratogen if present atlarge amounts or if it is present at unusual times. When applied exogenously during gastrulation,RA affects patterning of the anterior-posterior axis in many vertebrates (Alexandre et al., 1996).In such experiments, one consistent effect of RA is to alter the formation of the hindbrain and togenerally shift the fate of anterior structures toward more posterior fates. For example in thepresence of RA, mouse embryos vertebrae that would normally develop as cervical vertebraedevelop instead as thoracic vertebrae with ribs (Kalthoff, 2001). In zebrafish (Danio rerio),application of RA during the midgastrula stage caused a loss of defined brain regions and RAaddition at stages immediately prior to and after gastrulation showed additional effects on headdevelopment (Holder and Hill, 1991).In this experiment, we will investigate the effects of retinoic acid on development of the JapaneseMedaka (Oryzias latipes)\ Medaka embryos will be treated with varying concentrations of RAand phenotypes will be characterized according to gross morphology. ProcedureCAUTION! Retinoic acid is a powerful human teratogen. Use care and practice goodlaboratory procedures when