Complex cardiac defects after ethanol exposure during discrete cardiogenic events in zebrafish: prevention with folic acid

Fetal alcohol spectrum disorder (FASD) describes a range of birth defects including various congenital heart defects (CHDs). Mechanisms of FASD-associated CHDs are not understood. Whether alcohol interferes with a single critical event or with multiple events in heart formation is not known. RESULTS: Our zebrafish embryo experiments showed that ethanol interrupts different cardiac regulatory networks and perturbs multiple steps of cardiogenesis (specification, myocardial migration, looping, chamber morphogenesis, and endocardial cushion formation). Ethanol exposure during gastrulation until cardiac specification or during myocardial midline migration did not produce severe or persistent heart development defects. However, exposure comprising gastrulation until myocardial precursor midline fusion or during heart patterning stages produced aberrant heart looping and defective endocardial cushions. Continuous exposure during entire cardiogenesis produced complex cardiac defects leading to severely defective myocardium, endocardium, and endocardial cushions. Supplementation of retinoic acid with ethanol partially rescued early heart developmental defects, but the endocardial cushions did not form correctly. In contrast, supplementation of folic acid rescued normal heart development, including the endocardial cushions. CONCLUSIONS: Our results indicate that ethanol exposure interrupted divergent cardiac morphogenetic events causing heart defects. Folic acid supplementation was effective in preventing a wide spectrum of ethanol-induced heart developmental defects

Embryonic Ethanol Exposure Affects Early- and Late-Added Cardiac Precursors and Produces Long-Lasting Heart Chamber Defects in Zebrafish

Drinking mothers expose their fetuses to ethanol, which produces birth defects: craniofacial defects, cognitive impairment, sensorimotor disabilities and organ deformities, collectively termed as fetal alcohol spectrum disorder (FASD). Various congenital heart defects (CHDs) are present in FASD patients, but the mechanisms of alcohol-induced cardiogenesis defects are not completely understood. This study utilized zebrafish embryos and older larvae to understand FASD-associated CHDs. Ethanol-induced cardiac chamber defects initiated during embryonic cardiogenesis persisted in later zebrafish life. In addition, myocardial damage was recognizable in the ventricle of the larvae that were exposed to ethanol during embryogenesis. Our studies of the pathogenesis revealed that ethanol exposure delayed differentiation of first and second heart fields and reduced the number of early- and late-added cardiomyocytes in the heart. Ethanol exposure also reduced the number of endocardial cells. Together, this study showed that ethanol-induced heart defects were present in late-stage zebrafish larvae. Reduced numbers of cardiomyocytes partly accounts for the ethanol-induced zebrafish heart defects

Zebrafish as a Vertebrate Model System to Evaluate Effects of Environmental Toxicants on Cardiac Development and Function

Environmental pollution is a serious problem of the modern world that possesses a major threat to public health. Exposure to environmental pollutants during embryonic development is particularly risky. Although many pollutants have been verified as potential toxicants, there are new chemicals in the environment that need assessment. Heart development is an extremely sensitive process, which can be affected by environmentally toxic molecule exposure during embryonic development. Congenital heart defects are the most common life-threatening global health problems, and the etiology is mostly unknown. The zebrafish has emerged as an invaluable model to examine substance toxicity on vertebrate development, particularly on cardiac development. The zebrafish offers numerous advantages for toxicology research not found in other model systems. Many laboratories have used the zebrafish to study the effects of widespread chemicals in the environment on heart development, including pesticides, nanoparticles, and various organic pollutants. Here, we review the uses of the zebrafish in examining effects of exposure to external molecules during embryonic development in causing cardiac defects, including chemicals ubiquitous in the environment and illicit drugs. Known or potential mechanisms of toxicity and how zebrafish research can be used to provide mechanistic understanding of cardiac defects are discussed

Ethanol-induced Retinal Defects are Rescued by Retinoic Acid Supplement in Developing Zebrafish Embryos

poster abstractFetal Alcohol Spectrum Disorder (FASD) is caused by prenatal alcohol exposure, producing a spectrum of defects including facial abnormalities, sensory (visual and auditory) deficits, impaired fine motor skills and learning deficits including mental retardation. Our laboratory has used a zebrafish model for FASD that exposes embryos to ethanol during early development (midblastula transition through somitogenesis). Children diagnosed with FASD frequently show severe eye defects ranging from small eyes, underdeveloped optic nerve, and cataract. Zebrafish embryos exposed to ethanol showed defects similar to human eye birth defects. Presence of ethanol affected the differentiation of many retinal cell types including, retinal ganglion cells and photoreceptors. We hypothesize that ethanol may affect retinal patterning by competing with Retinaldehyde dehydrogenase (Raldh), reducing retinoic acid (RA) synthesis and signaling. Co-treatment of embryos with ethanol and 10-9 M RA could rescue the photoreceptor and retinal ganglion cell differentiation defects in the retina. RA plays a crucial role in the dorso-ventral patterning of the retina, and the enzymes involved in RA biosynthesis are expressed in the ventral retina during mid-somitogenesis stage. Our experiments showed that ethanol exposure during that critical time window when Raldh is expressed in the ventral retina causes severe defects in retinal cell specification. No defects were induced by ethanol exposure at the earlier stages. Presence of RA during photoreceptor differentiation could rescue ethanol-induced photoreceptor differentiation defects. Future work will dissect molecular mechanisms underlying ethanol defects, including retinoic acid-mediated eye development mechanisms. Determining the effects of ethanol exposure on retinal morphogenesis and differentiation will help identify potential therapeutic targets for ocular defects in this regrettably frequent birth defect syndrome

Zebrafish retinal stem cell differentiation mechanisms are disrupted by embryonic ethanol exposure

poster abstractPrenatal alcohol exposure can lead to a wide range of developmental abnormalities, which are included under the umbrella term fetal alcohol spectrum disorder (FASD). To understand the genesis of FASD defects, the zebrafish is important mechanistic animal model, particularly for retinal development. Previous work from our laboratory showed that ethanol treatment during gastrulation through somitogenesis in zebrafish embryos could recapitulate human ocular defects including microphthalmia, optic nerve hypoplasia, and photoreceptor defects. Ethanol-treated embryos showed increased retinal proliferation in the outer nuclear layer (ONL), inner nuclear layer (INL), and ciliary marginal zone (CMZ). Retinoic acid (RA) and folic acid (FA) co-supplementation rescued most ethanol-induced retinal defects, suggesting that nutrient deficiencies contribute to FASD. To better understand the genesis of ethanol-induced retinal cell differentiation defects, effects of ethanol exposure on retinal stem cell populations in the CMZ and Müller glial cell populations were examined. Ethanol treated retinas had an expanded CMZ, and a reduced expression domain for the cell cycle exit marker, cdkn1c. Ethanol treated retinas also showed reduced GFAP-positive Müller glial cells, which are a stem cell population in the central retina. At 72 hpf, the ONL of ethanol exposed fish showed few photoreceptors expressing terminal differentiation markers. Importantly, these poorly differentiated photoreceptors co-expressed the bHLH differentiation factor, neuroD, indicating that ethanol exposure produced immature and undifferentiated photoreceptors. Reduced differentiation along with increased progenitor marker expression and proliferation suggest cell cycle exit disruption due to ethanol exposure. Ethanol exposure severely disrupted Wnt and Notch signaling, which are critical for stem cell behavior and differentiation. These defects were rescued by Wnt signaling agonist, RA, and FA treatments. These results suggest ethanol disrupted retinal cell differentiation mechanisms. Further analysis of underlying molecular mechanisms will provide insight into the ethanol-induced retinal defects and potential therapeutic targets

Embryonic Ethanol Exposure Dysregulates BMP and Notch Signaling, Leading to Persistent Atrio-Ventricular Valve Defects in Zebrafish

Fetal alcohol spectrum disorder (FASD), birth defects associated with ethanol exposure in utero, includes a wide spectrum of congenital heart defects (CHDs), the most prevalent of which are septal and conotruncal defects. Zebrafish FASD model was used to dissect the mechanisms underlying FASD-associated CHDs. Embryonic ethanol exposure (3–24 hours post fertilization) led to defects in atrio-ventricular (AV) valvulogenesis beginning around 37 hpf, a morphogenetic event that arises long after ethanol withdrawal. Valve leaflets of the control embryos comprised two layers of cells confined at the compact atrio-ventricular canal (AVC). Ethanol treated embryos had extended AVC and valve forming cells were found either as rows of cells spanning the AVC or as unorganized clusters near the AV boundary. Ethanol exposure reduced valve precursors at the AVC, but some ventricular cells in ethanol treated embryos exhibited few characteristics of valve precursors. Late staged larvae and juvenile fish exposed to ethanol during embryonic development had faulty AV valves. Examination of AVC morphogenesis regulatory networks revealed that early ethanol exposure disrupted the Bmp signaling gradient in the heart during valve formation. Bmp signaling was prominent at the AVC in controls, but ethanol-exposed embryos displayed active Bmp signaling throughout the ventricle. Ethanol exposure also led to mislocalization of Notch signaling cells in endocardium during AV valve formation. Normally, highly active Notch signaling cells were organized at the AVC. In ethanol-exposed embryos, highly active Notch signaling cells were dispersed throughout the ventricle. At later stages, ethanol-exposed embryos exhibited reduced Wnt/β-catenin activity at the AVC. We conclude that early embryonic ethanol exposure alters Bmp, Notch and other signaling activities during AVC differentiation leading to faulty valve morphogenesis and valve defects persist in juvenile fish

Introducing Biology Undergraduates to Authentic Research through Grand Challenges in Global Health: Examining Environmental Factors that Influence the Development of Zebrafish Embryos

To increase student excitement and engagement in science, a course-based undergraduate research experience (CURE) has been introduced in the curriculum at IUPUI. In Fall 2013, original research projects investigating prenatal alcohol, nicotine and caffeine exposure effects on development of zebrafish embryos was introduced into the Introductory Biology K102 course. This research project was also a part of a new Themed Learning Community (TLC) at IUPUI called “From Molecules to Medicines” that examined grand challenges in global health. In documenting the developmental effects on zebrafish embryos, and designing new protocols to address student research questions, students gained experience with authentic research methods, laboratory techniques, microscopy, image analyses, statistical analyses, scientific writing and presentation skills. This project, especially in a freshman undergraduate lab setting, requires a new way of problem-solving, but greatly facilitates student excitement and engagement in science through the use of research-based high-impact practices for student success and persistence. To continue an inquiry-based lab on global health issues and to keep IUPUI biology curricula current with the rapid rise of bioinformatics, concepts of bioinformatics were introduced into the Cell Biology Laboratory K325 course in Spring, 2014. Students were allowed to work on their own investigatory projects to analyzed zebrafish microarray data to find genes affected after ethanol exposure. Students used NCBI/ Ensembl databases to retrieve the gene/protein sequences, and various freely available tools (GeneBank, Protein Data Bank, BLAST, ClustalW, ExPasy, Phylogenetic Tree) to investigate the evolutionary conservation of genes/proteins affected after ethanol exposure. Student learnt 3D-protein structure construction and observed how 3D-protein structure could change with single amino acid changes. Preliminary assessment indicates that students are gaining an understanding the web-based databases and tools and enjoying the investigatory nature of the lab exercises

Wnt Signaling in Zebrafish Fin Regeneration: Chemical Biology Using GSK3b Inhibitors

poster abstractBone growth can be impaired due to disease, such as osteoporosis, and Wnt signaling pathways regulate bone growth. The parathyroid hormone (PTH) is therapeutic for anabolic bone growth (bone building), which activates Wnt signaling, leading to bone growth. GSK3b (glycogen synthetase kinase 3 beta) protein inhibitors activate Wnt signaling, including in bone growth models. Our study utilized a zebrafish model system to study Wnt activated fin regeneration and bone growth. Wnt signaling is the first genetically identified step in fin regeneration, and bony rays are the main differentiated cell type in fins. Thus, zebrafish fin regeneration may be a useful model to study Wnt signaling mediated bone growth. Fin regeneration experiments were conducted using various concentrations of GSK3b inhibitor compound for different treatment periods and regenerative outgrowth was measured at 4 and 7 days post amputation. Experiments revealed continuous low concentration (5-6 nM) treatment to be most effective at increasing regeneration. Higher concentrations inhibited fin growth, perhaps by excessive stimulation of differentiation programs. In situ hybridization experiments were performed to examine effects of Gsk3b inhibitor on Wnt responsive gene expression. Initial experiments show temporal and spatial changes on individual gene markers following GSK3b inhibitor treatment. Additionally, confocal microscopy and immunofluorescence labeling data indicated that the Wnt signaling intracellular signal transducer, betacatenin, accumulates throughout Gsk3b inhibitor treated tissues. Finally, experiments are underway to quantify phosphohistone-3 staining in regenerating tissue to measure effects of Gsk3b inhibitor on cell proliferation. Together, these data indicate that bone growth in zebrafish fin regeneration is improved by activating Wnt signaling. Zebrafish Wnt signaling experiments provide good model to study bone growth and bone repair mechanisms, and may provide an efficient drug discovery platform

Effect of Curcuminoids in Turmeric on Developing Zebrafish Treated with Ethanol

poster abstractThis experiment was designed with the intention of determining whether turmeric could act as a rescue agent to prevent or mitigate the extent of Fetal Alcohol Spectrum Disorder (FASD) caused by early ethanol exposure using zebrafish as a model system. A range of turmeric concentrations were made from a stock solution of turmeric dissolved in ethanol (1mg turmeric in 5mL ethanol). The active agents in turmeric are the curcuminoids: Curcumin, Desmethoxycurcumin, and Bisdemethoxycurcumin. The curcuminoids concentration was estimated using liquid chromatography. These agents were present in the turmeric stock solution at the following concentrations: Bisdemethoxycurcumin: 36.6 +/- 0.1 ug/mL, Desmethoxycurcumin: 43.4 +/- 0.1 ug/mL, and Curcumin: 124.1 +/- 0.2 ug/mL. Untreated zebrafish embryos were placed in embryo medium, ethanol treated embryos in 100mM ethanol containing embryo medium, and turmeric co-supplemented medium with differing concentrations of turmeric. Since the turmeric stock solution was dissolved in ethanol, the concentration of ethanol was kept at a constant 100mM ethanol and the amount of turmeric solution added. The concentrations of the test plates were then based on this solution and made to be 100 mM ethanol and 1.16 uM curcuminoids, 100 mM ethanol and 1.74 uM curcuminoids, and 100 mM ethanol and 2.32 uM curcuminoids. The developing embryos were treated with the turmeric solution and/or ethanol during 2-24 hours post fertilization (hpf). These embryos were imaged at 72 hpf and their body length and eye diameter were measured. The embryos supplemented with curcuminoids showed a significant rescue effect on the body length and eye diameter compared to ethanol treated embryos. This indicates that the curcuminoids acted as a rescue agent to reduce the effects that are typical of FASD in developing zebrafish