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Intrauterine growth restriction (IUGR) is commonly observed in human pregnancies and can result in severe clinical outcomes. IUGR is observed in Fetal Alcohol Syndrome (FAS) fetuses as a result of alcohol (ethanol) exposure during pregnancy. To further understand FAS, the severe form of Fetal Alcohol Spectrum Disorder, we performed an extensive quantitative analysis of the effects of ethanol on embryo size utilizing our Xenopus model. Ethanol-treated embryos exhibited size reduction along the anterior-posterior axis. This effect was evident primarily from the hindbrain caudally, while rostral regions appeared refractive to ethanol-induced size changes, also known as asymmetric IUGR. Interestingly, some embryo batches in addition to shortening from the hindbrain caudally also exhibited an alcohol-dependent reduction of the anteriorhead domain, known as symmetric IUGR. To study the connection between ethanol exposure and reduced retinoic acid levels we treated embryos with the retinaldehyde dehydrogenase inhibitors, DEAB and citral. Inhibition of retinoic acid biosynthesis recapitulated the growth defects induced by ethanol affecting mainly axial elongation from the hindbrain caudally. To study the competition between ethanol clearance and retinoic acid biosynthesis we demonstrated that, co-exposure to alcohol reduces the teratogenic effects of treatment with retinol (vitamin A), the retinoic acid precursor. These results further support the role of retinoic acid in the regulation of axial elongation.
#128094 Canadian Institutes for Health Research, #316286 Manitoba Liquor & Lotteries, NA Wolfson Family Chair in Genetics, #668/17 Israel Science Foundation, #2013422 United States-Israel Binational Science Foundation, #2017199 United States-Israel Binational Science Foundation, #128094 CIHR
Figure 1. Natural length variation in the Xenopus laevis embryo population.Embryos were obtained from multiple fertilizations and all were allowed to develop to early tailbud stage (st. 30), when they were measured. (A) Schematic depiction of the different size parameters measured in the embryos. Size parameters: A, Whole embryo length; B, Forebrain-Midbrain length; C, Hindbrain-Trunk length; D, Eye anterior-posterior diameter; E, Dorsal-Ventral length. (B) Overall length (parameter A) distribution in normal Xenopus laevisembryos at st. 30. For each fertilization, the average length of all siblings in the clutch was calculated and for each embryo its relative length change (%) from the average was calculated and plotted. The 10thpercentile threshold was determined empirically by placing 10% of the overall mixed sample below the threshold based on the sample size (n). (C) Individual plotting of the five embryo clutches comprising the 5 biological replicates constituting the control sample in (B). The length change was calculated comparing each clutch to the overall average length calculated in (B).
Figure 2. Ethanol affects the length of the embryo in a concentration-dependent manner. Late blastula embryos were treated with increasing concentrations of ethanol (EtOH; 0.3%-0.9% vol/vol), DEAB (30 μM - 300 μM), or citral (30 μM - 60 μM). The embryos were allowed to develop to early tailbud stage (st. 30) and the length of each embryo was measured. For each batch (clutch) of embryos the 10th percentile was empirically determined for analysis of the treatments. (A) Embryos treated with increasing concentrations of ethanol. (B) Treatment with increasing concentrations of DEAB to inhibit retinoic acid biosynthesis. (C) Inhibition of retinoic acid production with increasing concentrations of citral. The percentage of embryos below the 10th percentile threshold is given above the X-axis. *, p<0.05; ****, p< 0.0001.
Figure 3. Ethanol induces several morphological malformations resembling inhibition of retinoic acid biosynthesis. Embryos were treated with DEAB, ethanol (EtOH) or citral at increasing concentrations. At early tailbud stage the embryos were analyzed for morphological malformations. (A-F) Treatments with DEAB and ethanol. (A) Control embryo. Embryos treated with 30 μM DEAB (B), 60 μM DEAB (C), 0.5% (vol/vol) ethanol (D), 1% ethanol (E) and 1.3% ethanol (F). To exemplify the size change, lines depicting the same parameters as in Fig. 1A were drawn on the control embryo (A) and then copied unto the treated embryos. (G-I) Citral treatment. (G) Control embryo. Embryos treated with citral 20 μM (H) or 40 μM. Also here a lines were drawn on the control embryo and copied unto the experimental embryos.
Figure 4. Ethanol and reduced retinoic acid prevent the normal elongation of the embryo. Xenopusembryos were treated with increasing ethanol (EtOH) concentrations (0.5%-13% vol/vol) or several DEAB concentrations (30 μM and 60 μM). At stage 30 different size parameters (Fig. 1A) were measured. (A) Whole embryo length. (B) Forebrain-Midbrain length. (C) Hindbrain-Trunk length. (D) Eye anterior-posterior diameter. (E) Dorsal-Ventral length. The percentage of embryos below the 10th percentile threshold is given above the X-axis. **, p<0.01; ***, p<0.001; ****, p< 0.0001.
Figure 5. Xenopus embryos can exhibit symmetric IUGR. Embryos of specific batches (clutches) exhibit symmetric IUGR in contrast to the majority of clutches that exhibit asymmetric IUGR as a result of ethanol (EtOH) exposure (Fig. 4). Whole embryo length determination (A), and forebrain-midbrain length (B) of embryos exhibiting symmetric IUGR. (C) Schematic depiction of Asymmetric and Symmetric IUGR. *, p<0.05; **, p<0.01; ****, p< 0.0001; ns, not significant.
Figure 6. The hindbrain - anterior spinal cord domain is the most sensitive to the alcohol exposure. Embryos treated with 0.5% or 0.9% ethanol (EtOH) from late blastula stages (st. 8.5) and analyzed during neurula stages (st. 17-18). The embryos were processed for in situhybridization to detect rhombomeres 3 and 5 (Egr2) and the posterior axis (MyoD). (A) Control embryo labeled for the distances measured. R3-R5, the distance between rhombomeres 3 and 5; R3-MyoD, the distance from rhombomere 3 to the rostral boundary of the MyoD expression; MyoD, the length of the MyoD expression domain. (B) Embryo treated with 0.5% ethanol where the gap from rhombomere to the MyoD expression is missing. (C) R3-R5 distances. (D) R3-MyoD distances. (E) MyoD length. All distances are shown as changes (%) from the average distance in the control sample. (F) Summary of the gap between R3 and MyoD in control and ethanol (EtOH) treated embryos. **, p<0.01; ***, p<0.001; ****, p< 0.0001; ns, not significant.
Figure 7. Reduced retinoic acid signaling affects embryonic length during gastrula and early neurula. Embryos were with 0.5% ethanol (EtOH) (A), 0.9% ethanol (B), 60 μM DEAB (C) or 50 μM citral (D). For different groups of embryos, the treatments were initiated at different developmental stages from mid-blastula (st. 8) to mid-neurula (st. 17). At st. 30 the embryos were analyzed for length changes. The percentage of embryos below the 10th percentile threshold is given above the X-axis. **, p<0.01; ***, p<0.001; ****, p< 0.0001; ns, not significant.
Figure 8. Ethanol and retinol compete in the developing embryo.Mid-blastula embryos were treated with ethanol (0.5%), retinol (30 μM) or a combination of both. Embryos were allowed to develop to early tailbud stages and then scored for the extent of their developmental malformations. (A) Control. (B) Ethanol treatment. (C) Retinol (vitamin A) exposure. (D) Ethanol and retinol combined treatment. (E) Summary of the phenotype distribution. *, p<0.05; **, p<0.01.
