Ethanol alters gene expression and cell organization during optic vesicle evagination

Abstract

Ethanol has been described as a teratogen in vertebrate development. During early stages of brain formation, ethanol affects the evagination of the optic vesicles, resulting in synophthalmia or cyclopia, phenotypes where the optic vesicles partially or totally fuse. The mechanisms by which ethanol affects the morphogenesis of the optic vesicles are however largely unknown. In this study we make use of in situ hybridization, electron microscopy and immunohistochemistry to show that ethanol has profound effects on cell organization and gene expression during the evagination of the optic vesicles. Exposure to ethanol during early eye development alters the expression patterns of some genes known to be important for eye morphogenesis, such as rx3/1 and six3a. Furthermore, exposure to ethanol interferes with the acquisition of neuroepithelial features by the eye field cells, which is clear at ultrastructual level. Indeed, ethanol disrupts the acquisition of fusiform cellular shapes within the eye field. In addition, tight junctions do not form and retinal progenitors do not properly polarize, as suggested by the mis-localization and down-regulation of zo1. We also show that the ethanol-induced cyclopic phenotype is significantly different to that observed in cyclopic mutants, suggesting a complex effect of ethanol on a variety of targets. Our results show that ethanol not only disrupts the expression pattern of genes involved in retinal morphogenesis, such as rx3 and rx1, but also disrupts the changes in cell polarity that normally occur during eye field splitting. Thus, ethylic teratology seems to be related not only to modifications in gene expression and cell death but also to alterations in cell morphology.

Keywords: ISH; MET; MHB; SEM; TUNEL; ZO-1; cell polarity; cyclopic mutants; eye specification; hours post-fertilization; hpf; in situ hybridization; mesenchymal–epithelial transition; midbrain–hindbrain boundary; morphogenesis; oep; one-eye pinhead; qRT-PCR; quantitative real-time polymerase chain reaction; somites stage; ss; standard error of mean; terminal deoxynucleotidyl transferase dUTP nick end labeling; zonula-occludens-1.

Fig. 1

Expression patterns of rx3, rx1 and six3 in embryos at 3 ss (a), 6 ss (b) and 18 ss (c). These genes show an altered expression (ethanol phenotype). Quantifications on the number of embryos that show control and ethanol phenotypes in each stage and treatment are shown in the graphs at the left. Scale bar = 100 μm.

Fig. 2

Expression pattern of otx2 (a–f), zic2 (g–l) and pax6 (m–r). None of these genes is altered in the anterior part of the embryo. pax6 expression is down-regulated in the region of the hindbrain and anterior spinal cord after exposure to ethanol (p–r). a: anterior; d: dorsal; l: lateral. Asterisk: diencephalon; black arrow: telencephalon; black arrow-head: midbrain; white arrow: midbrain–hindbrain boundary; white arrow-head: eye field; white line: hindbrain and anterior spinal cord. Scale bar = 100 μm.

Fig. 3

Expression pattern of emx1, wnt1 and wnt8 at different stages in control animals and in embryos exposure to ethanol. Scale bar = 100 μm.

Fig. 4

Cytoarchitecture of the eye field. At 3 ss retinal progenitors present big nuclei and circular shape (arrows in a), but not in the ethanol-treated embryos (b) which present several pyknotic nuclei (arrowhead in b). At 6 ss, retinal progenitors have fusiform morphologies only in control animals (arrows in c and d). From 10 ss onward, two optic vesicles are distinguishable only in un-treated animals (e–h). Retinal progenitors show an elongated morphology in control animals (arrows in i, k) compared to the circular shape in embryos exposed to ethanol (arrows in j, l), that only have them in the ventral part of the retina (arrowheads in l). d: dorsal; Di: diencephalon; EF: eye field; l: lateral; OV. Optic vesicle; T: telencephalon. Scale bar: a–h = 50 μm; i–l = 20 μm.

Fig. 5

Cell death assay by TUNEL at 75% epiboly (a, b), three (c, d), six (e, f) and 18 (g, h) somites. The number of positive cells (i) is higher in embryos exposed to ethanol at three, six and 18 somites both within the eye field (arrows) and in other regions of the anterior part (arrowheads). ^∗∗∗P < 0.0001.

Fig. 6

(a, b) Electron microscopy images from retinal precursor of 10-ss embryos. Retinal progenitors within the eye field display epithelial characteristics (a) with many well-organized junctions (arrow in inset in a). Exposed embryos present mitochondrias with aberrant morphologies (arrows in b), low density of ribosomes, big vacuoles (asterisks in b) and disorganized cell junctions (arrowheads in b). (c–f′) ZO-1 is located in the apical part of the cells at 6 (c, c′) and 10 ss (e, e′) and cells are oriented toward a mid axis. After exposure to ethanol, ZO-1 staining is dispersed and there is no organization of a mid axis (d, d′, f, f′). (g) qRT-PCR of zo1 expression at 10 ss. Exposure to ethanol down-regulates zo1 expression in a dose-dependent manner. d: dorsal, l: lateral. Scale bar: a = 2500 nm; inset in a, b = 500 nm; c–f′ = 50 μm. ^∗P < 0.05; ^∗∗0.05 > P > 0.001.

Fig. 7

Distribution pattern of ZO-1 in zebrafish mutants oep (a–b′), cyc (c–d′), tri (e–f′) and slb (g–h′). All of them show an aberrant accumulation of ZO-1, however the phenotype is qualitatively different from that observed in ethanol treated embryos. d: dorsal, l: lateral. Scale bar: a–h′ = 50 μm.