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The Xenopus homolog of glycogen synthase kinase-3, Xgsk-3, plays a major role in regulating the formation of the dorsal-ventral axis, most likely through effects on the mesoderm. To determine whether Xgsk-3 is involved in ectodermal patterning, Xgsk-3 was ectopically overexpressed in the presumptive ectoderm. This approach resulted in a dramatically expanded cement gland, which is due to early changes in cement gland specification at the anterior end of the embryo. Explant experiments were used to show that Xgsk-3 overexpression enhances the response of ectoderm to cement-gland-inducing signals from the mesoderm and to the intercellular signaling factor noggin. Expression of two other noggin-inducible genes, Xotx2 and XANF-2, was also expanded in whole embryos, while the expression of the epidermal marker, Xgbx-2, was eliminated. These results suggest that Xgsk-3 may play a role in anterior ectodermal patterning as a component of an intracellular pathway that regulates the ectodermal responsiveness to endogenous inducing signals.
FIG. 1. Ectopic Xgsk-3 expression causes cement gland expansion. Embryos were injected in one cell at the two-cell stage with Xgsk-3
RNA and cultured until stage 24. (A) Uninjected sides; dorsal is to the left and anterior is at the top. (B) Injected sides of embryos in (A);
dorsal is to the right. The sites of the endogenous cement glands are indicated (arrowheads).
FIG. 2. Cement gland expansion is limited to the anterior region of ectopic Xgsk-3 expression. Embryos were injected with Xgsk-3 and
b-galactosidase RNA in one cell at the two-cell stage, cultured until stage 20, and stained for b-galactosidase activity (red) and XAG-1
expression (purple). (A) Uninjected sides; dorsal is to the right and anterior is at the top. (B) Injected sides of the embryos in (A); dorsal is
to the left. Note that XAG-1 expression is limited to the anterior region of b-galactosidase activity. Occasional b-galactosidase expressing
cells on the uninjected side of the embryo indicate that the first cleavage does not always perfectly divide the embryo into left and right
halves.
FIG. 3. Ectopically expressed Xgsk-3 disrupts expression of anterior and lateral genes. Embryos were injected in one cell at the two-cell
stage with Xgsk-3FS (E) or Xgsk-3 (A–D, F–H) and b-galactosidase RNA, cultured until stage 13.5 (A) or stage 14 (B– H), and stained for
b-galactosidase activity and the indicated gene expression. (A and B) XAG-1 expression in embryos viewed from the dorsal side. Anterior
is facing to the left in (A) and to the right in (B), with the injected sides facing down. Note the presence of b-galactosidase activity and
the absence of XAG-1 expression within the neural plate region (arrowhead) in (B). Although not apparent in the photograph, the neural
folds, which mark the borders of the neural plate, are readily seen with a microscope. (C and D) Xotx2 expression on the uninjected (C)
and injected (D) sides of one embryo. Note anterior (arrow) and posterior (arrowhead) regions of expression in (C). In the embryo in (D),
only the anterior region of expression has expanded. (E and F) Anterior-lateral view of XANF-2 expression in embryos injected with Xgsk-
3FS (E) and Xgsk-3 (F) RNA. Note the appearance of anterior (arrow) and posterior (arrowhead) regions of expression in (E). In the embryo
in (F), both the anterior and posterior regions of expression have expanded on the injected side (facing down). (G and H) Xgbx-2 expression
on the uninjected (G) and injected (H) sides of one embryo. The epidermal expression is visible in (G). The neural domain of expression
is mostly hidden in this view by the neural folds. The pairs of embryos in (C and D) and (G and H) are oriented with dorsal at the top
and anterior facing the center. Staining for b-galactosidase activity is visible in the microscope although it is not visible in all photographs.
FIG. 4. Xgsk-3 enhances ectodermal response to mesoderm. Ectoderm was explanted from embryos previously injected with Xgsk-3FS
or Xgsk-3 RNA, cultured alone or in contact with dorsal mesoderm, and stained for XAG-1 expression. (A) Ectoderm from embryos injected
with Xgsk-3FS RNA, cultured alone. (B) Ectoderm from embryos injected with Xgsk-3 RNA, cultured alone. (C) Mesoderm from uninjected
embryos. (D) Ectoderm as in (A) cultured with mesoderm. (E) Ectoderm as in (B) cultured with mesoderm. The spot of stain in (C) is debris
stuck to the explant.
FIG. 5. Xgsk-3 enhances ectodermal responsiveness to noggin. Embryos were injected with Xgsk-3FS RNA (A), Xgsk-3 RNA (B), 37.5 pg of
noggin RNA in combination with Xgsk-3FS (C) or Xgsk-3 (D) RNA, or 150 pg of noggin RNA alone (E). Ectoderm was explanted, cultured, and
stained for XAG-1 expression. The diffuse pale purple color in some explants is background staining. One of three experiments is shown.
