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Shi Y, Li J, Chen C, Gong M, Chen Y, Liu Y, Chen J, Li T, Song W.
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BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: Our study demonstrates that prenatal alcohol exposure causes neural crest cell migration abnormality and 5-mehtyltetrahydrofolate could be beneficial for treating FASD.
Figure 1. Alcohol exposure impaired pigment and cranial cartilage formation. (A,C) Control embryos show normal amount and pattern of pigment cells. (B,D) Alcohol treated embryos develop less and punctate pigment cells. (E) Alcohol treated embryos at stage 45 exhibited truncated cranial cartilage. Compared to control embryos, both Meckel’s and palatoquadrate were absent from treated embryos and the ceratohyal cartilages were markedly smaller. (F) The alcohol exposure deteriorates cartilage formation. Ratio of small to total heads exhibit no significant difference between control (2.3% ± 1.5%, n = 190) and 0.5% alcohol condition (2.3% ± 0.7%, n = 214). 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 < 0.01). At each concentration, the experiments were repeated 3 times, and n represents the total embryos of triplicate assay. The values represent means ± SEM. p < 0.01, by student T test.
Shi et al. Molecular Brain 2014 7:67 doi:10.1186/s13041-014-0067-9
Figure 2. xposure to lower concentration of alcohol had no significant toxicity to neural crest development. We harvested the embryos at stage 16 for Whole-mount In Situ Hybridization (WISH). (A) Both boarder determinator gene Pax3, Zic1 and neural crest specification marker gene slug are kept intact. (B) Quantification of the effect of alcohol exposure on the expression of neural crest markers. (C) RT-PCR assays display expression of Msx1, Pax3, Zic1 and Slug are identical according to divergent alcohol treatment. (D,E) Alcohol less than 2% did not trigger abnormality of cell proliferation (labeled with pH3). Neural crest cell apoptosis was induced by 2% alcohol treatment as shown by activated caspase3. (normalized with beta actin).
Figure 3. Alcohol exposure disrupts neural crest migration. (A) Both slug and twist1 in situ hybridization, performing along with gradient alcohol treatment, displays neural crest migration is accordingly blocked by the increasing concentration of alcohol. (B,G) indicates 0.5% alcohol treatment still allows fairly migration of neural crest versus control embryos (A,F). 1.0% alcohol treatment deteriorate the egressing process of neural crest, however, there is perceivable migration occurring (C,H). Finally, both 1.5% and 2.0% appears completely freezing the migration (D,E,I,J).
Figure 4. 5-mehtyltetrahydrofolate improves alcohol-induced developmental abnormalities.(A) Homocysteine was significantly increased at stage 27 (from 25.5 ± 3.7 micromole/L /100embryos in control group to 41.8 ± 6.3 micromole/L /100embryos in alcohol group, P < 0.05), whereas the alteration is undetectable statistically at stage 16(23.5 ± 2.5 micromole/L /100embryos in control and 23.0 ± 2.3 micromole/L /100embryos in alcohol group, P > 0.05). Supplementation of 5MTHF significantly (P < 0.05) buffers the accumulation of endogenous homocysteine to 30.5 ± 2.3 micromole/L /100embryos at stage 27. (B,C) Injection of 5MTHF restored neural crest migration, and alleviated the effect of alcohol on neural crest migration. (D) Schematic diagram illustrating the xenografting experiments. (E-G) Alcohol exposure partially or completely blocked GFP-labeled neural crest xenografting migrating in living embryos. (H) Quantification of the GFP-labeled Neural crest migation upon gradient alcohol exposure and 5MTHF treatment.
