Characterization of fetal alcohol spectrum disorders (FASD) associated dental defects - insights from zebrafish (Danio rerio)

Abstract

Fetal alcohol spectrum disorders (FASD) describe a range of physical and behavioral anomalies caused by alcohol exposure during fetal development. The effects of FASD on craniofacial and dental formation remain under-investigated. Alcohol may interfere with the Wnt signaling pathways, which are essential for proper tooth development. Zebrafish serve as an ideal model organism for studying FASD. The objective of this study was to evaluate the effect of alcohol on zebrafish tooth development, analyzing tooth length, width, number, and shape at four time points: 15, 20, 25, and 30 days post-fertilization (dpf). The interaction between alcohol and Wnt signaling pathways using a Wnt agonist, lithium chloride (LiCl), a Wnt antagonist, WC59, and both in combination with alcohol is also explored. Tooth development was assessed using whole-mount cartilage and bone staining and imaging techniques, and the expression of Wnt10a and Wnt10b, essential for tooth development, was analyzed using in-situ hybridization. Results showed that alcohol-exposed zebrafish had significantly shorter tooth height and width than the control group at 15 dpf {(height- 58.29 ± 0.90 µm vs. 69.81 ± 0.39 µm), (width- 14.29 ± 1.24 µm vs. 22.73 ± 0.87 µm), P < 0.001)} and 20 dpf, {(height- 63.96 ± 1.56 µm vs. 72.40 ± 0.72 µm), (width- 22.35 ± 1.15 µm vs. 24.65 ± 0.61 µm), P < 0.001)} but not at 25 and 30 dpf. The number of teeth did not differ significantly between the two groups. LiCl treatment resulted in increased tooth length and width, while these metrics decreased in WC59 treatment group compared to the control at all time points (P < 0.001). Combining alcohol with LiCl or WC59 produced a significant reduction in tooth length and width compared to controls or alcohol-only treatment at all four time points (P < 0.001). Alizarin red-stained whole-mount zebrafish samples showed hypo-mineralized enamel tissues in treated samples at all time points compared to controls. This study highlights the impact of alcohol and its interaction with Wnt signaling pathway on tooth development and the importance of zebrafish in studying the phenotypic characteristics of fetal alcohol spectrum disorder.