Yelin R et al. (2007), Early molecular effects of ethanol during verte...

XB-ART-35324

Differentiation 2007 Jun 01;755:393-403. doi: 10.1111/j.1432-0436.2006.00147.x.

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Early molecular effects of ethanol during vertebrate embryogenesis.

Yelin R , Kot H , Yelin D , Fainsod A .

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Fetal alcohol spectrum disorder (FASD) is the combination of developmental, morphological, and neurological defects that result from exposing human embryos to ethanol (EtOH). Numerous embryonic structures are affected, leading to a complex viable phenotype affecting among others, the anterior/posterior axis, head, and eye formation. Recent studies have provided evidence suggesting that EtOH teratogenesis is mediated in part through a reduction in retinoic acid (RA) levels, targeting mainly the embryonic organizer (Spemann's organizer) and its subsequent functions. EtOH-treated Xenopus embryos were subjected to an analysis of gene expression patterns. Analysis of organizer-specific genes revealed a transient delay in the invagination of gsc- and chordin-positive cells that eventually reach their normal rostro-caudal position. Dorsal midline genes show defects along the rostro-caudal axis, lacking either their rostral (Xbra and Xnot2) or caudal (FoxA4b and Shh) expression domains. Head-specific markers like Otx2, en2, and Shh show abnormal expression patterns. Otx2 exhibits a reduction in expression levels, while en2 becomes restricted along the dorsal/ventral axis. During neurula stages, Shh becomes up-regulated in the rostral region and it is expressed in an abnormal pattern. These results and histological analysis suggest the existence of malformations in the brain region including a lack of the normal fore brain ventricle. An increase in the size of both the prechordal plate and the notochord was observed, while the spinal cord is narrower. The reduction in head and eye size was accompanied by changes in the eye markers, Pax6 and Tbx3. Our results provide evidence for the early molecular changes induced by EtOH exposure during embryogenesis, and may explain some of the structural changes that are part of the EtOH teratogenic phenotype also in FASD individuals.

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Species referenced: Xenopus laevis
Genes referenced: chrd en2 foxa4 gbx2.1 gbx2.2 gsc not otx2 pax6 shh tbx3 tbxt
GO keywords: eye development [+]

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	Fig. 1 The teratogenic effects of ethanol (EtOH). Control (A, C, E) and EtOH-treated (2.5% vol/vol) embryos (B, D, F) were analyzed at st. 41. (A, B) Lateral view of the whole embryos to ascertain the shortening induced by EtOH. (C, D) Lateral view of embryos at higher magnification to demonstrate the craniofacial malformations and the microphthalmia induced by EtOH. (E, F) Comparison of heads (dorsal view) to show the EtOH-induced microcephaly.
	Fig. 2 Delayed morphogenetic movements of organizer-derived cells as a result of ethanol (EtOH) exposure. EtOH-treated embryos were analyzed by in situ hybridization during mid-gastrula, late gastrula and early neurula stages in order to determine the fate of organizer-derived cells. (A–D) Control and EtOH-treated embryos hybridized with the gsc probe to study the migration of the prospective prechordal plate cells. (A, B) Embryos studied during midgastrula (st. 11) exhibiting the delay in rostral migration induced by EtOH. (C, D) By late gastrula stages (st. 12.5) the gsc signal continues to show a delay in migration in EtOH embryos compared with control embryos. The dotted lines mark the position of the dorsal blastoporal lip. (E–H) Localization of the final position of the gsc- and chordin-positive cells in EtOH-treated embryos (st. 14). (E) Control and (F) EtOH-exposed embryos studied with the gsc probe and a Gbx2 probe to mark the midbrain/hindbrain boundary (MHB; arrows). (G) Control and (H) EtOH-treated embryos hybridized with chordin and en2 probes to localize the position of the former relative to the midbrain/hindbrain boundary (arrows).
	Fig. 3 Abnormal Xnot2 expression following ethanol (EtOH) exposure. (A, B) Xnot2 expression at st. 12 following EtOH treatment. (A) Control and (B) EtOH-treated embryos analyzed by in situ hybridization with the Xnot2 specific probe. (C, D) Xnot2 and gsc expression following EtOH exposure. (C) Control and (D) EtOHtreated embryos were studied by double in situ hybridization with Xnot2 (magenta) and gsc (turquoise) probes. Elimination of the Xnot2 dorsal blastopore signal (magenta) allows the visualization of the gsc signal (turquoise). The insets are the same embryos at lower magnification. (E–H) Xnot2 downregulation by gsc overexpression (E, G) Control and (F, H) gsc overexpressing embryos were subjected to in situ hybridization analysis with the Xnot2 (E, F) and chordin (G, H) probes.
	Fig. 4 Ethanol (EtOH) affects midline genes along the rostro-caudal axis. (A, C, E, G) Control and (B, D, F, H) EtOH-treated embryos were subjected to in situ hybridization analysis of changes in midline gene expression during neurula stages (A, B) Embryos analyzed for changes in FoxA4b (FKH1) expression. (C, D) Embryos hybridized with the Shh-specific probe. (E, F) Embryos studied for changes in Xbra expression. (G, H) Analysis of Xnot2 expression during neurula stages.
	Fig. 5 The head domain is repressed by ethanol (EtOH) exposure. Embryos were treated with increasing concentrations of EtOH and allowed to develop to st. 26. Analysis of the head domain was performed by in situ hybridization with the head-specific gene, Otx2. (A) Control untreated embryo. (B) Embryo treated with 2% EtOH. (C) Embryo treated with 2.5% EtOH. Frontal views of the embryos.
	Fig. 6 The effect of ethanol (EtOH) on head structure formation. Embryos were treated with different concentrations of EtOH and allowed to develop to st. 36 (A–F, J–O) or st. 22 (G–I). The embryos were processed for in situ hybridization with probes specific for the en2 (A–F; Hemmati-Brivanlou et al., 1990b) and shh (G–O; Ekker et al., 1995a) genes. (A, D, G, J, M) Control untreated embryos. (B, E, H, K, N) Embryos treated with 2% EtOH. (C, F, I, L, O) Embryos treated with 2.5% EtOH. (A–C, G–I, M–O) Lateral views. (D–F, J–L) Dorsal view. Anterior is to the left. The arrows demarcate the prechordal mesoderm.
	Fig. 7 Ethanol (EtOH) affects forebrain and eye development. Embryos treated with EtOH were subjected to morphological (A, B), marker gene expression (C, D), and histological analysis (E, F). (A) Control and (B) EtOH-treated embryos (st. 26/27) were subjected to optical sagittal sectioning. Prosenc., prosencephalon; PCP, prechordal plate; notoch., notochord; purple arrow, forebrain ventricle; red arrows, prechordal plate width; white arrow, forebrain and spinal cord size. (C) Control and (D) EtOH-exposed embryos (st. 26/27) were subjected to double in situ hybridization with markers for the cement gland (XCG1, magenta), eye (Pax6, turquoise), midbrain/hindbrain boundary (MHB; en2, megenta), and rhombomeres 3 and 5 (Krox20, turquoise). (E) Control and (F) EtOHtreated embryos (st.24) were cross-sectioned to perform a histological analysis of the effects of EtOH on head development. The cement gland, prosencephalic, mesencephalic, and eye regions are marked.
	Fig. 8 The effect of ethanol (EtOH) on eye field development. Embryos were treated with increasing concentrations of EtOH. At st. 26 the embryos were fixed and processed for in situ hybridization with a probe specific for the Tbx3 gene. (A) Control embryo. (B) 1% EtOH (vol/vol). (C) 2% EtOH. (D) 2.5% EtOH. All embryos in lateral view, anterior to the left.
	Fig. 9 Microphthalmia and Pax6 down-regulation as a result of EtOH treatment. Control (A, C, E, G) and 2.5% ethanol (EtOH;vol/vol)-treated embryos (B, D, F, H) were allowed to develop to stages 26 (A–D), 34 (E, F) and 40 (G, H) and, subsequently, processed for in situ hybridization with a probe specific for the eye gene, Pax6. Panels (A, B, E–H) are lateral views, panels (C, D) are dorsal views. Anterior is to the left.
	Fig. 10 Pax6 is down-regulated by Shh overexpression. Embryos were injected with Shh mRNA and subjected to analysis of Pax6 expression by in situ hybridization at st. 32. (A) Control untreated embryo. (B) Embryo injected with Shh capped RNA..