5-mehtyltetrahydrofolate rescues alcohol-induced neural crest cell migration abnormalities
© Shi et al.; licensee BioMed Central Ltd. 2014
Received: 25 July 2014
Accepted: 29 August 2014
Published: 16 September 2014
Alcohol is detrimental to early development. Fetal alcohol spectrum disorders (FASD) due to maternal alcohol abuse results in a series of developmental abnormalities including cranial facial dysmorphology, ocular anomalies, congenital heart defects, microcephaly and intellectual disabilities. Previous studies have been shown that ethanol exposure causes neural crest (NC) apoptosis and perturbation of neural crest migration. However, the underlying mechanism remains elusive. In this report we investigated the fetal effect of alcohol on the process of neural crest development in the Xenopus leavis.
Pre-gastrulation exposure of 2-4% alcohol induces apoptosis in Xenopus embryo whereas 1% alcohol specifically impairs neural crest migration without observing discernible apoptosis. Additionally, 1% alcohol treatment considerably increased the phenotype of small head (43.4%± 4.4%, total embryo n = 234), and 1.5% and 2.0% dramatically augment the deformation to 81.2% ± 6.5% (n = 205) and 91.6% ±3.0% (n = 235), respectively (P < 0.05). Significant accumulation of Homocysteine was caused by alcohol treatment in embryos and 5-mehtyltetrahydrofolate restores neural crest migration and alleviates homocysteine accumulation, resulting in inhibition of the alcohol-induced neurocristopathies.
Our study demonstrates that prenatal alcohol exposure causes neural crest cell migration abnormality and 5-mehtyltetrahydrofolate could be beneficial for treating FASD.
Fetal alcohol spectrum disorders (FASD) results from maternal alcohol abuse and is featured by abnormal facial morphology, ocular anomalies, congenital heart defects, microcephaly and intellectual disability . Dysregulation of neural crest (NC) development has been contributing to the majority of malformations. Studies using numerous animal models reveal that alcohol can initiate neural crest cell apoptosis, and eventually develops FASD -.
NC cells are multipotent migratory cells originating at the ectoderm. After induction and specification, NC cells form many cell types ranging from the cranial bone, smooth muscle to peripheral and enteric neurons and glia -. NC development is tightly regulated by finely tuned gene regulatory network (GRN) and orchestrated hierarchically. NC induction is initialized at beginning of neurulation. Morphorgens such as Wnt, fibroblast growth factor (FGF), retinoic acid (RA) and bone morphogenetic protein (BMP) secreted from the paraxial mesoderm and epidermis regulates the expression of a group of transcription factors (Pax3, Zic1, Msx1) whereby defines the boarder of neural crest. Subsequently, these transcription factors stimulate the expression of the NC specification genes including FoxD3, Slug and Twist to determine the cell fate of NC ,. Once NC induction is accomplished NC cells undergo the epithelial-mesenchymal transition (EMT) and eventually migrate along predetermined trajectories.
Given NC induction and migration occurs sequentially ,, induction associated neurocristopathies can be identified to reflect migration abnormality. Alcohol appears to be able to affect NC development via either interfering NC cell migration or prompting cell apoptosis  that is indistinguishable from induction defects. Folate deficiency has long been known to be involved in NC defects. Abnormal folic acid level leads to dysfunction of NC migration ,. The disturbance of folic acid metabolism causes craniofacial malformation , and heart defects . Furthermore, studies showed that supplementation of folic acid successfully modulates alcohol-induced gene expression profiles and alleviates the developmental defects ,. However, the exact mechanism underlying on what specific developmental phase folic acid affects remains elusive.
In this study using Xenopus model system we investigated whether alcohol directly affected migration. We found that pre-gastrulation exposure of 2-4% alcohol induces developmentally non-specific apoptosis, whereas 1% alcohol specifically impairs neural crest migration without significant apoptosis, which consequently lead to neurocristopathies-like abnormalities. Furthermore, we showed that homocysteine, a molecular reported to affect neural crest migration , was elevated after alcohol exposure and 5-mehtyltetrahydrofolate (5MTHF) ameliorated endogenous homocysteine levels induced by alcohol exposure and dramatically rescued the impaired neural crest migration.
Results and discussion
Alcohol induces neurocristopathies in Xenopus leavis
Alcohol causes neither neural crest induction nor cell apoptosis at neural plate stage
Dysregulation of cell proliferation and apoptosis are implicated in alcohol-triggered neural crest deficiency ,,. However, results from different species are controversial and inconsistent . We examined this issue in Xenopus model system using immunohistochemistry with phosphohistone H3 (pH3) and activated caspase-3 antibodies to detect cell proliferation and apoptosis, respectively. The Xenopus embryos were incubated in alcohol-MBS buffer at stage 9 until harvest time at stage16. Alcohol exposure at 2% did not change the number of pH3 positive cells in the NC region at stage 16 (Figure 2D pink cells inside white dot line box). The cell apoptosis was not increased at stage 16 until the alcohol concentration over 2% as shown by activated-caspase 3 western blot assay (Figure 2E). Our data suggest that alcohol exposure (<2%) does not affect cell proliferation nor apoptosis in Xenopus neural crest induction.
Alcohol impairs NC cell migration
5MTHF rescues alcohol-induced neural crest migratory abnormality in Xenopus model
FASD has been largely attributed to dysregulation of NC development via either disruption of migration or cell apoptosis. In this study we found that alcohol treatment at 2% triggers apoptosis in Xenopus embryos, leading to neural crest deformations. Interestingly, we found that both 1.5% and 1.0% specifically inhibited NC migration without detectable apoptosis. These results indicate that alcohol preferentially affects the initiation of NC migration. Moreover, we found that the process of NC induction was not affected at 1.5% and 1.0% of alcohol treatment, suggesting that alcohol specifically interferes with NC migration. Our study suggest that human FADS could be more related to defective NC migration than apoptosis since human blood alcohol concentration is usually less than 0.5%.
FASD is a developmental disorder and the interaction between environment and gene plays a major role in the pathogenesis of FASD ,. Our study showed that 5-MTHF could potentially prevent the alcohol-induced developmental abnormalities. 5-MTHF plays a critical role in nucleotide synthesis and methylation processes. 5-MTHF is important to recycle homocysteine and synthesize S-adenosylmethionine (SAM). SAM is the main methyl donor providing methyl residue for the most of biological methylation reactions . Our previous work has indicated that modulating the metabolism of 5-MTHF affected the histone 3 lysine (H3K) methylation during neural crest development . Furthermore, it has been reported that the H3K methylation preferentially takes place at Snail and Twist1 enhancer, consequently controlling neural crest migration by altering transcriptional accessibility . Our study showed that alcohol resulted in accumulation of homocysteine at relatively late stage, indicating decreased level of 5-MTHF - during early neural crest development. Addition of 5-MTHF enables neural crest the capability of blocking the detrimental effects of alcohol on development. Clinical studies also showed significantly enhancement of serum homocysteine level upon alcohol consumption ,. Homocysteine has been known as a risk factor for neurocristopathies -. Alcohol appears to perturb neural crest migration by obstructing 5-MTHF absorption and increasing homocysteine. These detrimental effect could result in the abnormalities in FASD. Thus, modulation of one carbon cycle might be beneficial to prevention of FASD.
During embryonic development, alcohol targets many types of cell and/or organ -. Importantly, our observations are consistent with previous studies that alcohol causes NC migratory defectives via promoting apoptosis. Meanwhile, our results further indicate that the dysregulation of one carbon cycle during NC migration could be a novel mechanism underlying FASD. This discovery shed a light on the therapeutic possibility of folic acid. Our experiments were specifically designed to investigate toxicity of alcohol on NC development. Mesoderm plays a center role from the initiation to the migration of NC development, and tightly regulates the formation of whole body plan. Thus, further study is warranted to examine the role of mesoderm in NC development and its effect on FASD pathogenesis.
Our study demonstrate that prenatal alcohol exposure causes neural crest cell migration abnormality and 5-mehtyltetrahydrofolate could be beneficial for treating fetal alcohol spectrum disorders.
The experimental procedures for in vitro fertilization, manipulation and microinjection of the Xenopus embryos, as well as for the in situ hybridization of whole mount embryo were previously described . The cartilage staining was carried out using Alcian blue 8GX according to protocol of Dr. Richard Harland's lab (http://tropicalis.berkeleyedu/home/gene_expression/cartilage-stain/alcian.html) as well as described  and the in situ probes for Zic1, Pax3 Slug and Twist gene expression were described previously . Microinjection was performed using the PLI-1 Pico-injector (Harvard Apparatus) equipped with the MK-1 micromanipulator (Singer Instruments).
Reverse transcription - polymerase chain reaction (RT-PCR)
To test the effect of alcohol on neural crest induction, RT-PCR was carried out using whole embryos at the onset of gastrulation (stage9) until stage 16. H4 was used as a loading control. The primers used for PCR were: Pax3: 5′-CAGCCGAATTTTGAGGAGCAAAT-3′ and 5′-GGGCAGGTCTGGTTCGGAG TC-3′; Snail2: 5′ -TCC- CGCACTGAAAATGCCACGATC -3′ and 5′- CCGTCCTAA- AGATGAAGGGTATCCTG -3′. The primers for Msx1 were used as described . Please simply describe the procedure.
Embryo immunohistochemistry and western blot
Embryos were collected at stage 16 and fixed with paraformaldehyde. The frozen samples were sectioned in 10 μm and the sections were stained with 1:200 Anti-phospho-histone H3 (Millipore 16-657) to detect cell proliferation and 300 nM DAPI (Life Technologies D1306) for nuclear conterstain. Whole embryos at stage 16 were lysed with 1%triton-X100 in PBS containing a protease inhibitor cocktail (Roche) and the samples were subject to Western blot analysis with activated caspase3 antibody(Abcam 13847) to check cell apoptosis.
One hundred Xenopus embryos were collected at either stage 16 and 27 and homogenized in 500 μl of lysis buffer [1%triton-X100 in PBS containing a protease inhibitor cocktail (Roche)]. The samples were centrifuge at 12000 g for 10 min at 4°C. After centrifugation, samples were separated into 3 phases; the lower sediment, intermediate water phase (which contained homocysteine) and upper lipid phase. The intermediate water phase was carefully collected without any lipid contamination. The homocysteine concentration was determined by an enzymatic method (produced by Zhongyuan Bio. Ltd China) on an automatic analyzer (Olympus AU 2700). The linear range of Hcy measurement is from 3 μM to 50 μM.
All experiments were repeated at least 3 times, and the P value were calculated with T-TEST.
YS, JL, CC, TL, and WS conceived and designed the experiments; YS, JL, and CC performed the experiments; YS, JL, YL, and WS analyzed the data; MG and YC contributed reagents/materials/analysis tools; YS and WS wrote the paper. All authors read and approved the final manuscript.
We would like to thank Dr. Bingyu Mao for the experimental reagents. This work was supported by the National Natural Science Foundation of China (NSFC) Grants 81102519 (to Y.S.), 81200878 (J.L.), 81161120498 (T.L.), China Postdoctoral Science Foundation funded project 2012 M511914 (Y.S.), Chongqing Science and Technology Committee Grant cstc2012jjA0147 (Y.S.), and Canadian Institutes of Health Research (CIHR) Operating Grant TAD-117948 (W.S). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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