Zebrafish fetal alcohol syndrome model: effects of ethanol are rescued by retinoic acid supplement

Abstract

This study was designed to develop a zebrafish experimental model to examine defects in retinoic acid (RA) signaling caused by embryonic ethanol exposure. RA deficiency may be a causative factor leading to a spectrum of birth defects classified as fetal alcohol spectrum disorder (FASD). Experimental support for this hypothesis using Xenopus showed that effects of treatment with ethanol could be partially rescued by adding retinoids during ethanol treatment. Previous studies show that treating zebrafish embryos during gastrulation and somitogenesis stages with a pathophysiological concentration of ethanol (100mM) produces effects that are characteristic features of FASD. We found that treating zebrafish embryos with RA at a low concentration (10(-9)M) and 100mM ethanol during gastrulation and somitogenesis stages significantly rescued a spectrum of defects produced by treating embryos with 100mM ethanol alone. The rescued phenotype that we observed was quantitatively more similar to embryos treated with 10(-9)M RA alone (RA toxicity) than to untreated or 100mM ethanol-treated embryos. RA rescued defects caused by 100mM ethanol treatment during gastrulation and somitogenesis stages that include early gastrulation cell movements (anterior-posterior axis), craniofacial cartilage formation, and ear development. Morphological evidence also suggests that other characteristic features of FASD (e.g., neural axis patterning) are rescued by RA supplement.

Figure 1

Ethanol treatment of zebrafish embryos during gastrulation and somitogenesis stages produces a FASD phenotype that was largely rescued by including retinoic acid during ethanol treatment. Dorsal (left column) and lateral (right column; dorsal is up) views of 4 dpf living larvae were imaged using a steromicroscope; anterior is left on each image. Control embryos were untreated (first, top row); treated with 100 mM ethanol (second and third row from top; note FASD phenotype is variable); treated with 100 mM ethanol and 10⁻⁹ M retinoic acid (fourth row from top); and treated with 10⁻⁹ M retinoic acid alone (fifth, bottom row) from 3 hpf until 24 hpf (see Materials and Methods). At 24 hpf, ethanol and/or retinoic acid treatments were discontinued, and embryos were incubated with normal embryo medium until 4 dpf. Asterisks and arrows indicate the swim bladder. Abbreviations: ed, edematous pericardium; ey, eye; fin, pectoral fin; and ov, otic vesicle.

Figure 2

Ethanol treatment of zebrafish embryos during gastrulation and somitogenesis stages produces craniofacial defects that were improved by including retinoic acid during ethanol treatment. (A.) Dorsal views (anterior is left) of 5 dpf alcian blue stained larvae were imaged using a steromicroscope. Embryos were untreated in control embryos (top panel), and treated with 100 mM ethanol (second and third panels from top), 100 mM ethanol and 10⁻⁹ M retinoic acid (third panel from top), and 10⁻⁹ M retinoic acid alone (bottom panel) from 3 hpf until 24 hpf (see Materials and Methods). At 24 hpf, ethanol and/or retinoic acid treatments were discontinued, and embryos were incubated with normal embryo medium until 5 dpf. These 5 dpf larvae were processed for alcian blue staining to label craniofacial cartilages. White asterisks mark the ethmoid cartilage measured (in other embryos) for Table I. The ethmoid cartilage cannot be seen in the more severely affected ethanol treated embryo (second panel from top), and consequently, when we could not detect the cartilage, we did not include these embryos in our measurements. Note that the ethmoid cartilage extends beyond the hyoid arch (the most anterior structure in the ventral jaw cartilages). (B.) Scatter plot representation of ethmoid cartilage width variation in embryos treated from 3 hpf until 24 hpf (during gastrulation and somitogenesis) with 100 mM ethanol, 10⁻⁹ M retinoic acid, or 100 mM ethanol and 10⁻⁹ M retinoic acid together, and compared to untreated control embryos. Ethmoid plate cartilage width was measured in alcian blue stained 4.5 dpf larvae (see Table I for statistical analysis).

Figure 3

Scatter plot representation of ethmoid cartilage width variation in embryos treated from 3 hpf until 24 hpf (during gastrulation and somitogenesis) with 100 mM ethanol, 10⁻⁹ M retinoic acid, or 100 mM ethanol and 10⁻⁹ M retinoic acid together, and compared to untreated control embryos. Ethmoid plate cartilage width was measured in alcian blue stained 4.5 dpf larvae (see Table I for statistical analysis).

Figure 4

Ethanol treatment of zebrafish embryos during gastrulation and somitogenesis stages reduces neural axis patterning (particularly forebrain) and reduces hatching gland formation (a prechordal plate derivative), which was improved by including retinoic acid during ethanol treatment. Lateral views (anterior is left) of 30 hpf living embryos were imaged using DIC microscopy. Embryos were untreated in control embryos (top panel), and treated with 100 mM ethanol (second and third panels from top, note reduced forebrain, white asterisks, and reduced hatching gland, white arrows, which was variable), 100 mM ethanol and 10⁻⁹ M retinoic acid (fourth panels from top), and 10⁻⁹ M retinoic acid alone (bottom panel) from 3 hpf until 24 hpf (see Materials and Methods). At 24 hpf, ethanol and/or retinoic acid treatments were discontinued, and embryos were incubated with normal embryo medium until 30 dpf, anesthetized and imaged. Abbreviations: ey, eye; fb, forebrain; hb, hindbrain; hg, hatching gland; mid, midbrain; and ov, otic vesicle. In the four lower panels, asterisks indicate forebrains, and the hatching glands are indicated by arrows.