Ethanol exposure during mid-blastula transition changes the gene expression in zebrafish embryos. (A) Quantitative RT-PCR assays comparing transcript levels of sox2, her7, and dlc after ethanol treatment. Average fold change in expression was calculated from at least 3 independent experiments, with samples analyzed in triplicate. Samples were normalized to transcript levels for rsp15, and fold change for ethanol treated embryos was calculated by normalizing control levels to 1. (B–G) Whole mount in situ hybridization showed reduced expression of sox2 (B,C), dlc (D,E) and dld (F,G) in E100 embryos (C,E,G) compared to control (B,D,F).

Ethanol induced gene expression changes during zygotic genome activation. (A) Venn diagram shows overlapping of ethanol dysregulated genes identified in the Affymetrix GeneChip microarray analysis of control and ethanol treated embryos at 4.5 hpf with potential Sox2 targets and the genes differentially expressed in quadruple knockdown of SoxB1 factors, which includes Sox2 (Soxb1 QKD targets). (B) Twenty five of the ethanol-dysregulated transcription factors showing the enrichment of their targets in our ethanol-dysregulated gene set. (C) Transcription factor-target gene network visualizing using cytoscape shows interactions among the transcription factors (gold circles) and their target gene (blue circles) in our dataset. Ethanol dysregulated targets of Sox2 are co-shared by other dysregulated transcription factors.

Ethanol induced epiboly and gastrulation defect was partially rescued by sox2 mRNA injection. (A–D) Bright field images showed reduced epiboly progression after ethanol exposure (B; double arrow), which was rescued by sox2 mRNA injection (D). (E) Graph shows the percentage of epiboly progression in control, ethanol-treated, sox2 mRNA injected, and sox2 mRNA + ethanol-treated injected embryos (see text for statistics). (F–I) In situ hybridization detecting ntl expression shows dorsal forerunner cells closely associated to the germ band in control, embryo (F), sox2 mRNA injected (H), and ethanol-treated + sox2 mRNA injected (I) embryos, and a dramatic separation and fragmentation of the dorsal forerunner cells from the germ band and from each other in the ethanol treated embryo (G).

Ethanol induced gene expression changes was reversed by sox2 mRNA injection. (A–D) Whole mount ISH shows reduced dld expression after ethanol exposure, which was like control in sox2 mRNA injected and sox2 mRNA injected plus ethanol-treated embryos. (E) Quantitative PCR showed downregulation of sox2, her7, and dlc transcripts in the ethanol-treated embryos, which increased in the sox2 mRNA injected plus ethanol-treated embryos.