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
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
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
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..