Ethanol treatment causes cyclopia. Embryos were treated at the dome/30% epiboly stage with 2.4% ethanol for 3 h and grown to the 48-h stage. (A) Untreated control embryo. (B) Ethanol-treated embryo with separate, but more closely spaced eyes. (C to E) Ethanol-treated embryos with increasing severity of cyclopia. Embryos are shown in frontal views, dorsal up.

Ethanol treatment impairs tail development. Embryos were treated from the dome/30% epiboly stage with 2.4% ethanol for 3 h and then grown for 5 days in the absence of ethanol. (A) Untreated control embryo. (B to D) Cyclopic embryos generated by ethanol treatment showing varying degrees of impaired tail development.

Expression of shh, axl, and nk2.2 in the ventral brain is impaired by ethanol treatment. (A to C) Expression of shh in control embryo (A) and ethanol-treated embryos (B, C). (D to F) Expression of axl in control embryo (D) and ethanol-treated embryos (E, F). (G to I) Expression of nk2.2 in control (G) and ethanol-treated embryos (H, I). Embryos are at the 24-h stage. Arrowheads in A to C indicate the positions of the mid-hindbrain boundary. Orientation of embryos is anterior to the left and dorsal up.

Anteroposterior patterning of the brain is not affected by ethanol treatment. (A to C) pax-6 mRNA expression in untreated control (A) and ethanol-treated embryos (B, C). pax-6 expressions in the dorsal diencephalon (d) and hindbrain are indicated by arrowheads. (D to F) pax-2 expression in control (D) and ethanol-treated embryos (E, F). pax-2 expression in the optic stalks and ventral eye (arrowhead with dot) is progressively lost with increasing severity of cyclopia. In contrast, pax-2 expression in the posterior midbrain (arrowhead) and in the hindbrain is unaffected even in severely affected embryos. Orientation of embryos is anterior to the left; view onto dorsal (A, B) and lateral views (C to F).

Ethanol treatment impairs migration of prechordal plate cells. (A to D) Control embryo (A) and ethanol-treated embryos (B to D) at the 100% epiboly stage. Embryos were simultaneously stained with gsc (g) and pax-2 (p) probes. In most cases, gsc-expressing cells formed a cluster which was retarded in its anterior migration (B, C). Less frequently, the gsc expression domain (g) was split in the ethanol-treated embryo (D). (E to H) Control and ethanol-treated embryos stained with gsc and pax-2 antisense probes (blue) and anti-Ntl antibody (brown). The expression domains of gsc (g), pax-2 (p), and Ntl (N) are indicated; gsc expression in G is out of the plane of focus. Open triangles in F and H indicate planes of sections shown in Figs. 7B and 7A, respectively. Orientation of embryos is anterior up and view is from the dorsal aspect.

Aberrantly located, gsc-expressing cells form a distinct cell cluster and do not coexpress Ntl in the posterior axis of ethanol-treated embryos. Transverse sections through embryos labeled with gsc probe (blue, g) and anti-Ntl antibody (brown, n). Plane of section is through the tailbud (A) or through the hindbrain (B) as indicated by open triangles in Figs. 6H and 6F, respectively. Orientation of sections is dorsal up. Scale bar, 15 μm.

Expression of anterior midline markers in ethanol-treated embryos. (A and B) Control (A) and ethanol-treated embryos hybridized to hgg-1 (hg) and pax-2 (p) probes. (C and D) otx-2 expression in control (C) and ethanol-treated embryos (D) stained with otx-2 probe. Arrow indicates otx-2 expression in the posteriorly displaced prechordal plate in the ethanol-treated embryo (D). (E to G) Control (E) and ethanol-treated embryos (F, G) labeled with isl-1 (i) and pax-2 (p) probes. (H to L) Control (H) and ethanol-treated embryos stained with hlx-1 (h) and pax-2 probes (p). hlx-1 expression is impaired anterior to the pax-2 expression domains, but unlike hgg-1 (B) and isl-1 (C) hlx-1 transcripts are not detectable posterior to the pax-2 stripes (L, arrowhead). (M to P) Control (M) and ethanol-treated embryos (N to P) stained with shh (s) and pax-2 probes (p). Ethanol-treated embryos show progressive loss of anterior shh expression, while expression of shh posterior to the pax-2 stripes is unaffected in treated embryos. Embryos are at the 100% epiboly stage and are shown in dorsal views with anterior up.

Cyclopic embryos lack correctly positioned hatching glands. Ethanol-treated embryos at the 24-h stage were simultaneously stained for hgg-1 (red) and shh (blue) expression; hgg1 is specifically expressed in hatching glands. (A) Ethanol-treated embryo with narrowly spaced eyes have hatching glands over the yolk (arrows). Some hgg1-expressing cells were incorrectly positioned under the forebrain (open arrow). (B) Cyclopic embryo which lacks the wild-type pattern of hgg-1 expression on the yolk; hatching glands are instead found at the level of the forebrain and midbrain (arrows). Note that the anterior-most expression of shh in the hypothalamus is missing in this embryo (compare arrowheads marking anterior limit of shh expression in A and B). (C) Profoundly cyclopic embryo which lacks shh expression in the fore- and hgg-1-expressing cells are located in the trunk in this embryo (arrow). Orientation of embryos is anterior to the left and dorsal up. Arrowheads, anterior limits of shh expression.

Forskolin induces cyclopia but does not impair the migration of the prechordal plate. (A and B) Embryos (36 h old) treated with 100 mg/ml forskolin at the dome stage for 3 h with narrower eyes (A) or cyclopia (B). (C and D) Control (A) and forskolin-treated embryo at the 100% epiboly stage labeled with gsc (g) and pax-2 (p) probes. Orientation of embryos is frontal view, dorsal up (A and B); and dorsal view, anterior up (C and D).

Ectopic activation of axial (HNF-3β) in response to misexpression of shh is not affected by ethanol treatment. (A and B) Embryos (24 h old) injected with shh mRNA at the one- to two-cell stage and then treated with ethanol (A) or left untreated (B). (C) Uninjected control embryo. When compared to a control embryo (C), shh-injected embryos (A, B) have ectopic expression of axial (HNF-3β) in the anterior neural tube in the presence or absence of ethanol treatment (arrowheads). Orientation of embryos is lateral view with dorsal up and anterior to the left.