Common congenital anomalies: Environmental causes and prevention with folic acid containing multivitamins.

Congenital anomalies, congenital defects, or birth defects are significant causes of death in infants. The most common congenital defects are congenital heart defects (CHDs) and neural tube defects (NTDs). Defects induced by genetic mutations, environmental exposure to toxins, or a combination of these effects can result in congenital malformations, leading to infant death or long-term disabilities. These defects produce significant mortality and morbidity in the affected individuals, and families are affected emotional and financially. Also, society is impacted on many levels. Congenital anomalies may be reduced by dietary supplements of folic acid and other vitamins. Here, we review the evidence for specific roles of toxins (alcohol, cigarette smoke) in causing common severe congenital anomalies like CHDs, NTDs, and ocular defects. We also review the evidence for beneficial effects for dietary supplementation, and highlight gaps in our knowledge, where research may contribute to additional benefits of intervention that can reduce birth defects. Extensive discussion of common severe congenital anomalies (CHDs, NTDs, and ocular defects) illustrates the effects of diet on the frequency and severity of these defects. Birth Defects Research (Part C) 108:274-286, 2016. © 2016 Wiley Periodicals, Inc.

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.

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

BACKGROUND: 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.

Another Swim in the Extensive Pool of Zebrafish Research.

The zebrafish has gained utility in modeling biomedical phenomena for discovery research [...].

Behavioral Effects of the Mixture and the Single Compounds Carbendazim, Fipronil, and Sulfentrazone on Zebrafish (Danio rerio) Larvae.

Pesticides are often detected in freshwater, but their impact on the aquatic environment is commonly studied based on single compounds, underestimating the potential additive effects of these mixtures. Even at low concentrations, pesticides can negatively affect organisms, altering important behaviors that can have repercussions at the population level. This study used a multi-behavioral approach to evaluate the effects of zebrafish larvae exposure to carbendazim (C), fipronil (F), and sulfentrazone (S), individually and mixed. Five behavioral tests, thigmotaxis, touch sensitivity, optomotor response, bouncing ball test, and larval exploratory behavior, were performed to assess potential effects on anxiety, fear, and spatial and social interaction. Significant changes were observed in the performance of larvae exposed to all compounds and their mixtures. Among the single pesticides, exposure to S produced the most behavioral alterations, followed by F and C, respectively. A synergistic effect between the compounds was observed in the C + F group, which showed more behavioral effects than the groups exposed to pesticides individually. The use of behavioral tests to evaluate pesticide mixtures is important to standardize methods and associate behavioral changes with ecologically relevant events, thus creating a more realistic scenario for investigating the potential environmental impacts of these compounds.

Zebrafish cadherin-11 participates in retinal differentiation and retinotectal axon projection during visual system development.

BACKGROUND: Cadherins orchestrate tissue morphogenesis by controlling cell adhesion, migration and differentiation. Various cadherin family members are expressed in the retina and other neural tissues during embryogenesis, regulating development of these tissues. Cadherin-11 (Cdh11) is expressed in mesenchymal, bone, epithelial, neural and other tissues, and this cadherin was shown to control cell migration and differentiation in neural crest, tumor and bone cells. Our previous studies characterized Cdh11 expression and function in zebrafish. RESULTS: Here, we report effects of Cdh11 loss-of-function on visual system development using morpholino oligonucleotide knockdown methods. Cdh11 is expressed in the retina and lens during retinal differentiation. Cdh11 loss-of-function produced defects in retinal differentiation and lens development. Cdh11 loss-of-function also reduced retinotectal axon projection and organization, consistent with known Cdh11 function in cell migration. CONCLUSION: Cdh11 expression in the developing visual system and Cdh11 loss-of-function phenotype illustrates the critical role for differential cadherin activity in visual system differentiation and organization.

Essential Oils Produce Developmental Toxicity in Zebrafish Embryos and Cause Behavior Changes in Zebrafish Larvae.

Essential oils have gained significant popularity in various industries due to their biological properties, but their potential toxic effects on living organisms have been poorly investigated. This study aimed to evaluate the effects of lemongrass, thyme, and oregano essential oils on zebrafish embryos and larvae as animal models. Embryos were exposed to different concentrations of essential oils, and various endpoints were assessed, including epiboly, mortality (LC50), morphometry, and behavioral changes. All three essential oils reduced epiboly, affecting embryonic development. LC50 values were calculated for lemongrass (3.7 µg/mL), thyme (14.4 µg/mL), and oregano (5.3 µg/mL) oils. Larvae exposed to these oils displayed morphological defects, including growth reduction, spinal deformation, pericardial edema, eye size reduction, and reduced swim-bladder inflation. Morphometric analysis confirmed reduced larval length at higher oil concentrations. Essential-oil exposure altered zebrafish larval swimming behavior, with lemongrass oil reducing dark-cycle activity and oregano oil increasing light-cycle activity, suggesting neurodevelopmental toxicity. These findings illustrate the adverse effects of these oils on zebrafish embryos and larvae and reveal essential-oil toxicity, indicating careful use should be considered, particularly during pregnancy.

Ethanol Effects on Early Developmental Stages Studied Using the Zebrafish.

Fetal alcohol spectrum disorder (FASD) results from prenatal ethanol exposure. The zebrafish (Danio rerio) is an outstanding in vivo FASD model. Early development produced the three germ layers and embryonic axes patterning. A critical pluripotency transcriptional gene circuit of sox2, pou5f1 (oct4; recently renamed pou5f3), and nanog maintain potency and self-renewal. Ethanol affects sox2 expression, which functions with pou5f1 to control target gene transcription. Various genes, like elf3, may interact and regulate sox2, and elf3 knockdown affects early development. Downstream of the pluripotency transcriptional circuit, developmental signaling activities regulate morphogenetic cell movements and lineage specification. These activities are also affected by ethanol exposure. Hedgehog signaling is a critical developmental signaling pathway that controls numerous developmental events, including neural axis specification. Sonic hedgehog activities are affected by embryonic ethanol exposure. Activation of sonic hedgehog expression is controlled by TGF-ß family members, Nodal and Bmp, during dorsoventral (DV) embryonic axis establishment. Ethanol may perturb TGF-ß family receptors and signaling activities, including the sonic hedgehog pathway. Significantly, experiments show that activation of sonic hedgehog signaling rescues some embryonic ethanol exposure effects. More research is needed to understand how ethanol affects early developmental signaling and morphogenesis.

Neural Activity Correlates With Behavior Effects of Anti-Seizure Drugs Efficacy Using the Zebrafish Pentylenetetrazol Seizure Model.

Approximately 30% of patients with epilepsy do not achieve adequate seizure control through current anti-seizure drugs and treatment methods. Therefore, a critical need exists to efficiently screen anti-seizure drugs to enhance our ability to tailor treatment protocols and improve patient outcomes. The zebrafish pentylenetetrazol (PTZ) seizure model has become an increasingly popular screening paradigm for novel anti-seizure compounds. However, previous research using this model was variable due to differing experimental methods. Here, we present a method that was optimized to improve reliability and reproducibility in our laboratory using this PTZ model to develop a more robust screening of anti-seizure drugs comparing behavior and neural activity. Our behavior assay, spanning 90 min using 10 mM PTZ on 7 days post fertilization zebrafish, provides a broad window to observe anti-seizure drug efficacy. To compare our method with previously published data, we tested carbamazepine, lamotrigine, and topiramate, which have been tested in previous PTZ zebrafish assays. In addition, we assessed the candidate anti-seizure compound GS967, which has not been previously tested in the zebrafish seizure model. We examined the efficacy of anti-seizure drugs by acute administration concurrent with PTZ application and by pretreatment prior to exposure with PTZ. Pretreatment permitted us to examine potential neuroprotection and determine whether treatment time affects anti-seizure drugs' responses. As independent validation of anti-seizure drugs' effects, we evaluated whether the anti-seizure drug efficacy in the behavioral assay correlated with neural activity measurements, using electroencephalogram (EEG) and calcium signaling using GCaMP. There was no significant difference in the reduction of PTZ-induced seizure behavior activity between the pretreatment groups and acute treatment groups. Acute treatment with anti-seizure drugs in the EEG and GCaMP assays from 15 to 30 min post-anti-seizure drug exposure revealed consistent results between behavioral, EEG, and GCaMP assays for two of the three anti-seizure drugs. Lamotrigine only reduced neural activity (EEG and GCaMP assays). Carbamazepine, topiramate, and GS967 reduced activity in all three assays. The findings show that EEG and GCaMP assays largely correlate with the behavior findings, helping us connect physiological and behavior responses to anti-seizure drug and better assess anti-seizure drug efficacy.

Elf3 deficiency during zebrafish development alters extracellular matrix organization and disrupts tissue morphogenesis.

E26 transformation specific (ETS) family transcription factors are expressed during embryogenesis and are involved in various cellular processes such as proliferation, migration, differentiation, angiogenesis, apoptosis, and survival of cellular lineages to ensure appropriate development. Dysregulated expression of many of the ETS family members is detected in different cancers. The human ELF3, a member of the ETS family of transcription factors, plays a role in the induction and progression of human cancers is well studied. However, little is known about the role of ELF3 in early development. Here, the zebrafish elf3 was cloned, and its expression was analyzed during zebrafish development. Zebrafish elf3 is maternally deposited. At different developmental stages, elf3 expression was detected in different tissue, mainly neural tissues, endoderm-derived tissues, cartilage, heart, pronephric duct, blood vessels, and notochord. The expression levels were high at the tissue boundaries. Elf3 loss-of-function consequences were examined by using translation blocking antisense morpholino oligonucleotides, and effects were validated using CRISPR/Cas9 knockdown. Elf3-knockdown produced short and bent larvae with notochord, craniofacial cartilage, and fin defects. The extracellular matrix (ECM) in the fin and notochord was disorganized. Neural defects were also observed. Optic nerve fasciculation (bundling) and arborization in the optic tectum were defective in Elf3-morphants, and fragmentation of spinal motor neurons were evident. Dysregulation of genes encoding ECM proteins and matrix metalloprotease (MMP) and disorganization of ECM may play a role in the observed defects in Elf3 morphants. We conclude that zebrafish Elf3 is required for epidermal, mesenchymal, and neural tissue development.

The Genius of the Zebrafish Model: Insights on Development and Disease.

The zebrafish is an outstanding and inexpensive vertebrate model system for biomedical research [...].

Zebrafish (Danio rerio) larvae show behavioral and embryonic development defects when exposed to opioids at embryo stage.

Opioid abuse continues to plague society, and in recent years, there has been an epidemic, leading to increased addiction and death. It is poorly understood how prenatal opioid use affects the lives of children. The aim of this work was to evaluate the effect of early embryonic codeine or morphine exposure in zebrafish (Danio rerio), examining gastrulation progression (epiboly), teratogenic effects, mortality and locomotor behavior response to light/dark cycles. Zebrafish embryos were exposed to codeine or morphine (designated C or M) at 1, 5 or 10 mg/L (designated 01, 05 or 10, respectively) from 3 to 24 h postfertilization (hpf) or from 3 to 48 hpf (designated -24 or - 48 for 1 or 2 days of exposure, respectively). The C10-24, C01-48, C05-48 and C10-48 groups showed significantly smaller eyes than control larvae at 7 days postfertilization (dpf). Locomotor behavior of control larvae in light/dark cycles showed greater swimming time and distance in dark cycles. Two-day codeine exposure produced strong effects, showing no significant response due to light/dark cycles in distance moved. Morphine exposed groups showed similar effects as observed in 2-day codeine exposed groups, showing less large movement activity and also no significant difference between inactive duration in response to light/dark cycles. In conclusion, we observed low teratogenic effects and mortality effects. Animals exposed to high levels and higher exposure times of opioids were hypoactive, relative to controls, in the dark period. Future studies will be needed to understand the neural defects producing behavior changes.

Marijuana and Opioid Use during Pregnancy: Using Zebrafish to Gain Understanding of Congenital Anomalies Caused by Drug Exposure during Development.

Marijuana and opioid addictions have increased alarmingly in recent decades, especially in the United States, posing threats to society. When the drug user is a pregnant mother, there is a serious risk to the developing baby. Congenital anomalies are associated with prenatal exposure to marijuana and opioids. Here, we summarize the current data on the prevalence of marijuana and opioid use among the people of the United States, particularly pregnant mothers. We also summarize the current zebrafish studies used to model and understand the effects of these drug exposures during development and to understand the behavioral changes after exposure. Zebrafish experiments recapitulate the drug effects seen in human addicts and the birth defects seen in human babies prenatally exposed to marijuana and opioids. Zebrafish show great potential as an easy and inexpensive model for screening compounds for their ability to mitigate the drug effects, which could lead to new therapeutics.

Folic acid reduces the ethanol-induced morphological and behavioral defects in embryonic and larval zebrafish (Danio rerio) as a model for fetal alcohol spectrum disorder (FASD).

The objective of this work was to determine whether folic acid (FA) reduces the embryonic ethanol (EtOH) exposure induced behavioral and morphological defects in our zebrafish fetal alcohol spectrum disorder (FASD) model. Teratogenic effects, mortality, the excitatory light-dark locomotion (ELD), sleep (SL), thigmotaxis (TH), touch sensitivity (TS), and optomotor response (OMR) tests were evaluated in larvae (6-7 days post-fertilization) using four treatment conditions: Untreated, FA, EtOH and EtOH + FA. FA reduced morphological defects on heart, eyes and swim bladder inflation seen in EtOH exposed fish. The larvae were more active in the dark than in light conditions, and EtOH reduced the swimming activity in the ELD test. EtOH affected the sleep pattern, inducing several arousal periods and increasing inactivity in zebrafish. FA reduces these toxic effects and produced more consistent inactivity during the night, reducing the arousal periods. FA also prevented the EtOH-induced defects in thigmotaxis and optomotor response of the larvae. We conclude that in this FASD model, EtOH exposure produced several teratogenic and behavioral defects, FA reduced, but did not totally prevent, these defects. Understanding of EtOH-induced behavioral defects could help to identify new therapeutic or prevention strategies for FASD.

Protective effects of quercetin, polydatin, and folic acid and their mixtures in a zebrafish (Danio rerio) fetal alcohol spectrum disorder model.

Protective effects of quercetin (QUE), polydatin (POL), and folic acid (FA) and their mixtures were tested using zebrafish to model fetal alcohol spectrum disorder in this study. Zebrafish embryos were exposed to 150 mM ethanol for 6 or 22 h and co-treated with QUE, POL, FA, and their mixtures (37.5-100.0 µM). Epiboly progression, teratogenic effects, and behavior were evaluated. Ethanol exposure reduced epiboly, and FA and QUE protected against these ethanol-induced defects. POL did not reduce epiboly defects. The mixture QUE + FA showed a possible antagonistic effect. The observed teratogenic effects were similar in all ethanol exposed groups. QUE, FA and QUE + POL reduced the percentage of affected animals, but treatments did not eliminate teratogenic effects. Behavioral measurements were divided into small (between 4 and 8 mm/s) and high swimming activity (>8 mm/s). All experimental groups displayed a reduction in small swimming activity as compared to control and ethanol groups when exposed to bright light. Additionally, larvae exposed to ethanol were more inhibited than control, not showing a habituation period (after 60 min of experiment) in high swimming activity. Chemical treatments like QUE and POL reduced behavioral defects induced by ethanol exposure. In conclusion, this study presents new evidence that QUE, POL, FA and their mixtures partially protected epiboly, teratogenic, and behavioral defects induced by ethanol exposure. QUE, FA and QUE + POL were more effective in reducing these defects than the other studied compounds and mixtures.

Embryonic ethanol exposure alters expression of sox2 and other early transcripts in zebrafish, producing gastrulation defects.

Ethanol exposure during prenatal development causes fetal alcohol spectrum disorder (FASD), the most frequent preventable birth defect and neurodevelopmental disability syndrome. The molecular targets of ethanol toxicity during development are poorly understood. Developmental stages surrounding gastrulation are very sensitive to ethanol exposure. To understand the effects of ethanol on early transcripts during embryogenesis, we treated zebrafish embryos with ethanol during pre-gastrulation period and examined the transcripts by Affymetrix GeneChip microarray before gastrulation. We identified 521 significantly dysregulated genes, including 61 transcription factors in ethanol-exposed embryos. Sox2, the key regulator of pluripotency and early development was significantly reduced. Functional annotation analysis showed enrichment in transcription regulation, embryonic axes patterning, and signaling pathways, including Wnt, Notch and retinoic acid. We identified all potential genomic targets of 25 dysregulated transcription factors and compared their interactions with the ethanol-dysregulated genes. This analysis predicted that Sox2 targeted a large number of ethanol-dysregulated genes. A gene regulatory network analysis showed that many of the dysregulated genes are targeted by multiple transcription factors. Injection of sox2 mRNA partially rescued ethanol-induced gene expression, epiboly and gastrulation defects. Additional studies of this ethanol dysregulated network may identify therapeutic targets that coordinately regulate early development.

The Glycogen Synthase Kinase-3ß Inhibitor LSN 2105786 Promotes Zebrafish Fin Regeneration.

The Wnt pathway has been shown to regulate bone homeostasis and to influence some bone disease states. We utilized a zebrafish model system to study the effects of a synthetic, orally bioavailable glycogen synthase kinase-3ß (GSK3ß) inhibitor LSN 2105786, which activates Wnt signaling during bone healing and embryogenesis. GSK3ß inhibitor treatment was used to phenocopy GSK3ß morpholino oligonucleotide (MO) knockdown in zebrafish embryos. Human and zebrafish synthetic mRNA injection were similarly effective at rescue of GSK3ß MO knockdown. During caudal fin regeneration, bony rays are the first structure to differentiate in zebrafish fins, providing a useful model to study bone healing. Caudal fin regeneration experiments were conducted using various concentrations of a GSK3ß inhibitor, examining duration and concentration dependence on regenerative outgrowth. Experiments revealed continuous low concentration (4-5 nM) treatment to be more effective at increasing regeneration than intermittent dosing. Higher concentrations inhibited fin growth, perhaps by excessive stimulation of differentiation programs. Increased Wnt responsive gene expression and differentiation were observed in response to GSK3b inhibitor treatment. Activating Wnt signaling also increased cell proliferation and osteoblast differentiation in fin regenerates. Together, these data indicate that bone healing in zebrafish fin regeneration was improved by activating Wnt signaling using GSK3b inhibitor treatment. In addition, caudal fin regeneration is useful to evaluate dose-dependent pharmacological efficacy in bone healing, various dosing regimens and possible toxicological effects of compounds.

Cadherin-7 function in zebrafish development.

Cadherin cell adhesion molecules play crucial roles in vertebrate development. Most studies have focused on examining the functions of classical type I cadherins (e.g., cadherin-2) in the development of vertebrates. Little information is available concerning the function of classical type II cadherins (e.g., cadherin-7) in vertebrate development. We have previously shown that cadherin-7 mRNA exhibits a dynamic expression pattern in the central nervous system and notochord in embryonic zebrafish. To gain insight into the role of cadherin-7 in the formation of these structures, we analyzed their formation in zebrafish embryos injected with cadherin-7-specific antisense morpholino oligonucleotides (MO). Notochord development was severely disrupted in MO-injected embryos, whereas gross defects in the development of the central nervous system were not detected in MO-injected embryos. Our results thus demonstrate that cadherin-7 plays an important role in the normal development of the zebrafish notochord.

Retinal Wnt signaling defect in a zebrafish fetal alcohol spectrum disorder model.

Fetal alcohol spectrum disorder caused by prenatal alcohol exposure includes ocular abnormalities (microphthalmia, photoreceptor dysfunction, cataracts). Zebrafish embryos exposed to ethanol from gastrulation through somitogenesis show severe ocular defects, including microphthalmia and photoreceptor differentiation defects. Ethanol-treated zebrafish had an enlarged ciliary marginal zone (CMZ) relative to the retina size and reduced Müller glial cells (MGCs). Ethanol exposure produced immature photoreceptors with increased proliferation, indicating cell cycle exit failure. Signaling mechanisms in the CMZ were affected by embryonic ethanol exposure, including Wnt signaling in the CMZ, Notch signaling and neurod gene expression. Retinoic acid or folic acid co-supplementation with ethanol rescued Wnt signaling and retinal differentiation. Activating Wnt signaling using GSK3 inhibitor (LSN 2105786; Eli Lilly and Co.) restored retinal cell differentiation pathways. Ethanol exposed embryos were treated with Wnt agonist, which rescued Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. Our results illustrate the critical role of Wnt signaling in ethanol-induced retinal 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.