Figure 7A, B Microscope images of E10 neural tube sections with the indicated treatments were stained with antibodies against MARS (green) and N-Hcy (red). Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n=3) and are presented in (B). C, D Double immunofluorescent staining (C) for MARS (green) and N-Hcy (red) in E10 heart sections. Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n=3) and are presented in (D).Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.E Goss morphology of E10.5 ATRA-treated and untreated mice embryos. The ATRA-treated embryo exhibits an open neural tube (indicated by white arrows) compared to littermate controls. Scale bar: 500 μm.F Examples of normally developed and maldeveloped mice hearts: in the normal heart (i), the ink travelled from the right ventricle (RV) into the pulmonary trunk (PT); however, in ATRA-treated group hearts, ink travelled into the aorta (Ao) (ii, transposition of great arteries (TGA)) or both the PT and Ao (iii, double outlet right ventricle (DORV)). Scale bar: 1000 μm.G Cardiac malformations were analysed by hematoxylin and eosin (H&E) staining of heart sections from E14.5 mice. The cardiac defects observed in ATRA treated mice are as follows: Ventricular septal defect (VSD) and single ventricle defects (SVD). Heart compartments are marked: RA: right atria, RV: right ventricle, LA: left atria, LV: left ventricle. Scale bar: 1000 μmH Total protein N-Hcy levels in maternal mice liver homogenates were determined and quantified in correlation to the corresponding prevalence of NTDs in affected foetuses (i). Western blot was used to detect N-Hcy levels (ii). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.I Neural tube tissues at E10 were homogenized from litters of untreated, ATRA-treated, and ATRA+AHT co-treated pregnant mice. Neural tube homogenates of each group were mixed from 6 embryos. Endogenous SOD1 and SOD2 were affinity purified from the homogenates. N-Hcy levels and the specific activities of each purified SOD1 and SOD2 were determined. The SOD activities of the untreated group were set at 100%; error bar indicates assay replicates (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.J Superoxide (a), apoptosis (b), and Wnt signalling (c, d) were over-activated in ATRA-treated mice neural tubes and restored by AHT or NAC treatment. (a) Whole mount DHE staining of E10.5 mice embryos. Scale bar: 500 μm. Neural tube defect (white arrows) and heart (green arrow) regions show especially higher fluorescence intensities. (b) TUNEL (green) staining detected apoptotic cell death in E8.5 neural tube (E) sections. MARS (red). Scale bar: 300 μm. (c, d) IHC staining for β-catenin (green) in E8.5 (c) and E10 (d) mice neural tube sections. Scale bar: 300 μm (c), 150 μm (d). DAPI (blue).K Average (n=3) DHE fluorescence intensities were quantified relative to the untreated group. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.L Quantification of TUNEL-positive cells Three sections from three embryos were pooled for analysis. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.M Quantification of β-catenin fluorescence intensities in experiment J (b) and J (c). The average (n=3) β-catenin level in the untreated group was set at 100%. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.N Hcy-treated eggs exhibited NTDs phenotypes at embryonic stage 8 (E8). A normal embryo (a) and neural tube defect phenotypes (b, c) are shown. Exencephaly (white arrows) and spina bifida (blue arrows) are indicated. Scale bar: 500 μm.O Total protein N-Hcy levels of 72 h chicken embryo homogenates from Hcy and Hcy+AHT-treated groups were detected by western blot.P Whole mount DHE staining of 72 h chicken embryos from untreated, Hcy-, Hcy+AHT-, and Hcy+NAC-treated groups. Scale bar:2000 μm. Q Quantification of DHE fluorescence intensities in (P) (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.