|
Fig. 1. Maximal sensitivity to EtOH exposure during late blastula/early gastrula. The window of sensitivity for EtOH treatment was determined. Embryos were exposed to 2.5% EtOH from different developmental stages onwards and studied at stage 36. The embryos were incubated in a medium without EtOH (A) or placed in 2.5% EtOH at stages 8.5 (B), 11 (C), 12 (D), 13 (E) and 18 (F).
|
|
Fig. 2. Spemann's organizer is an early target of EtOH. Organizer-specific gene expression was studied in EtOH-treated embryos. Control (A, C, E, G, I) and EtOH-treated (B, D, F, H, J) embryos were analyzed for organizer specific gene expression at st. 10.5. The expression of chordin (A, B), gsc (C, D), Otx2 (E, F), Xnot2 (G, H) and Xbra (I, J) in EtOH-treated embryos is shown.
|
|
Fig. 3. The effect of EtOH on convergence-extension. Convergence-extension movements and elongation in EtOH-treated embryos were studied in exogastrulae and explanted dorsal marginal zones. Control (A) and EtOH-treated (B) exogastrulae and control (C) and EtOH-treated (D) explanted DMZs.
|
|
Fig. 4. Opposite effects of EtOH and RA on Hox and gsc expression. (A) Hox gene expression was studied at stage 13 by RT-PCR in embryos treated with EtOH (2.5%) or RA (1 μM). (B–D) In situ hybridization with the gsc-specific probe was used to corroborate the opposed effects of EtOH and RA. Changes in the expression pattern of gsc (st. 11) were determined in the control (B), EtOH-(C) and RA-treated (D) embryos.
|
|
Fig. 5. EtOH and RA have opposed effects on gene expression. (A) Quantitative real-time PCR was employed to study the changes in gsc, Otx2 and chordin expression at st. 10.5 as a result of EtOH (2.5%), RA (1 μM) and Citral (60 μM) treatment. P < 0.05. (B) Quantitative real-time PCR analysis of Cyp26 expression as a result of EtOH and RA treatment. Whole embryos (grey) or dorsal marginal zone explants (black) were processed for RNA extraction at st. 10.5. P < 0.005. (C) Embryos were injected with the RAREZ, RA reporter plasmid, and subsequently treated with EtOH and RA. Quantitation of the reporter plasmid activity was performed (st. 10.5–11) using a chemiluminiscent substrate for ß-galactosidase. P < 0.0005.
|
|
Fig. 6. Citral affects organizer-specific gene expression. In order to reduce the endogenous RA levels, embryos were treated with citral (60 μM). The embryos were processed for in situ hybridization with gsc (A, B), Otx2 (C, D), chordin (E, F) and Xnot2 (G, H) specific probes. Control untreated sibling embryos are marked.
|
|
Fig. 7. RA metabolism by Cyp26 overexpression results in changes in organizer-specific gene expression. Active RA levels were reduced by Cyp26 mRNA injection. At st. 10.5, the embryos were processed for in situ hybridization with chordin (A, B) and Otx2 (C, D) specific probes. Control untreated sibling embryos are marked.
|
|
Fig. 8. Retinoic acid signaling in Spemann's organizer. The detection of RA signaling was performed using the RAREZ or RAREGFP reporter plasmids. (A–C) Embryos injected with the RAREZ plasmid and stained for ß-galactosidase activity at stages (A) 13, (B) 30 and (C) 10.5. (D) RAREZ transgenic embryo at st. 11.5 stained for ß-galactosidase activity. (E) Transgenic embryo with the RAREGFP plasmid during early/mid gastrula stages. (F) Same embryo as in (E) illuminated by epifluorescence and normal illumination to demonstrate the location of the GFP signal. The dashed line marks the position of the dorsal lip of the blastopore. (G) Quantitation of the RA signaling using the RAREZ reporter plasmid. At the onset of gastrulation, the DMZ and VMZ regions were explanted from embryos injected with the RAREZ plasmid and were processed for quantification of the ß-galactosidase activity at st. 10.5–11. The control samples were whole embryos extracts. P < 0.015.
|
|
Fig. 9. EtOH can rescue the teratogenic effects of ROL and RAL. The rescue assays included phenotypic, gene expression and reporter plasmid activity assays. (A–D) A combined phenotypic and gene expression assay was performed at st. 33. Embryos were treated with: (A) control, (B) 70 μM ROL, (C) 1.5% EtOH and (D) 70 μM ROL together with 1.5% EtOH. The embryos were processed for in situ hybridization with probes for en2, Pax6 and XAG1. The insets are enlargements of the head region. (E–H) Rescue analysis during gastrulation based on the gsc expression pattern. The embryos were studied at st. 10.5 after treatment with (E) control, (F) 70 μM ROL (G) 1.5% EtOH and (H) 70 μM ROL and 1.5% EtOH. (I–L) EtOH can rescue the teratogenic effects of RAL. Embryos were studied at st. 33 for head phenotype and the expression of the en2, Pax6 and XAG1 markers. (I) control, (J) 5 μM RAL, (K) 1.5% EtOH and (L) 5 μM RAL and 1.5% EtOH. (M) Embryos were injected with the RAREZ plasmid, treated with ROL (150 μM), EtOH (1.5% or 2.5%), or a combination of both. The determination of the ß-galactosidase activity was performed at st. 10.5–11. P < 0.0001.
|
