November 15, 1991;
Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center.
Expression cloning from a pool of gastrula
cDNAs identified the Wnt family member Xwnt-8
as having dorsal axis-inducing activity in Xenopus embryos. Microinjected Xwnt-8
mRNA was able to rescue the development of a dorsally complete anterior
axis in embryos ventralized by exposure to UV light. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres at the 32-cell stage
. Vegetal blastomeres receiving Xwnt-8
mRNA contributed progeny not to the induced dorsal axis, but to the endoderm
, a result consistent with Xwnt-8
causing cells to act as a Nieuwkoop center (the vegetal-inducing component of normal dorsal axis formation), rather than as a Spemann organizer
(the induced dorsal marginal zone
component that directly forms the dorsal mesoderm
, which is normally expressed ventrally in midgastrula and neurula
embryos, appears to mimic, when injected, maternally encoded dorsal mesoderm
-inducing factors that act early in development.
[+] show captions
Figure 1. Size-Fractionated Poly(A)’ RNA from LiCl Embryos and N-CAM Induction
(A) Size-fractionated poly(A)t RNA from LiCl embryos. Sixty micrograms of poly(A)t RNA from LiCl embryos was size fractionated on a lO%-30%
sucrose gradient in the presence of methylmercuric hydroxide. One-twentieth of each of the 16 fractions collected was electrophoresed on a
formaldehyde-containing agarose gel. Nylon filter blots were made from the gel and probed with a “P end-labeled deoxythymidine 17-mer. An
autoradiograph of the resulting blot is shown.
(6) N-CAM induction by size-fractionated LiCl embryo poly(A)t RNA. Four-cell stage UV-ventralized embryos were microinjected with approximately
10 ng of size-fractionated poly(A)+ RNA. Total RNA was prepared from pooled embryos (32-62 per pool) at the tailbud stage. The RNA equivalent
of 2.5 embryos was electrophoresed on agarose/formaldehyde gels. Nylon filter blots were probed both for N-CAM and for EFl-a mRNAs. RNA
prepared from UV-ventralized embryos microinjected with RNA from fractions 7-9 is shown. Also shown is RNA from noninjected UV-ventralized
and normal embryos.
Figure 2. Dorsal Axis Rescue by Xwnt-8
UV-ventralized embryos were microinjected with increasing amounts
of Xwnt-8 encoding mRNA at the 4-cell stage. Representative tailbud
stage embryos are shown. (A) Normal embryo (no UV treatment or
injection). (B) Uninjected UV-ventralized embryo. (C) UV-ventralized
embryo injected with water alone. (D-G) UV-ventralized embryos microinjected
with 1, 10, 100, and loo0 pg of Xwnt-g-encoding mRNA,
respectively. Open arrow in (D) indicates posterior dorsal fin. Closed
arrows in (F) and (G) indicate enlarged cement glands.
Figure 3. Northern Slot Analysis of Developmentally Staged Normal,
LiCI-. and W-Treated Embryos
RNA was prepared from ten embryos from the indicated stages and
treatments. The RNA from 2.5 embryos was loaded on each lane. An
autoradiograph of a filter hybridized with random-primed Xwnt-8 and
EFl-a probes is shown.
Figure 4. In Situ Hybridization to Normal, LiCI-, and UV-Treated Embryos
Plain arrows indicate the edge of the blastocoel; arrowheads indicate the dorsal lip of the blastopore (or the lip of the blastopore for LiCl and UV
embryos); open arrows show the anterior limit of the archenteron.
(A) Stage 8 blastula; no staining.
(6) Stage 9-10 blastula. Dorsal is at the top. Nuclei around the marginal zone are stained. The dorsal quadrant lacks detectable staining (limits
of staining region shown with bars). Some background staining is evident in the blastocoel of (B) and (D) (see Harland, 1991).
(C) Stage 11 gastrula. Dorsal is at the top. Staining is limited to the ventral side in the marginal zone. Lateral views of such embryos are consistent
with the sectioned embryo presented in Christian et al. (1991 b).
(D) Stage 12 late gastrula, lateral view. The anterior limit of the archenteron is indicated. Staining is absent from the yolk plug and endoderm, but
otherwise extends in two “wings” around the closing blastopore.
(E) Stage 14 early neurula, lateral view. The blastocoel collapses during neurulation. Staining is limited to the posterior ventral tissue.
(F) Stage 16 neurula, dorsal view. Head is to the left. Arrowhead points to the remnant of the blastopore. Photograph is in the plane of the notochord,
which extends between the arrowhead and the open arrow. Staining is excluded from the mesoderm below the neural plate, but residual staining
is widespread in the posterior ventral mesoderm. Two patches of staining (one of which is marked with a square), are evident lateral to the anterior
(G) Head of stage 36 tadpole. Staining cells are visible next to the x.
(H) Stage 11 gastrula from LiCI-treated batch of embryos. The blastopore (arrowhead) closes vigorously in such embryos, leading to a puckered
appearance. No staining is evident (compare with [Cl).
(I) Vegetal view of a stage 11 UV-treated gastrula. The blastopore appears late in UV embryos. Staining surrounds the marginal zone of this embryo
(compare with [Cl).
Figure 5. Xwnt-8/B-Galactosidase mRNA Coinjection
(A-C) Animal (tier 1) (A), marginal (tier 2 or 3) (S), and vegetal (tier 4) (C) blastomeres of normal stage 6 embryos were microinjected with 0.5 ng
of f%galactosidase mRNA.
(D-G) Injections of mRNAs into UV-ventralized stage 6 embryos. (D) Marginal blastomeres with 0.5 ng of 6-galactosidase mRNA. (E) Animal
blastomeres with 0.5 ng of P-galactosidase plus 10 pg of Xwnt-6 mRNAs. (F) Marginal blastomeres with 0.5 ng of 6-galactosidase plus 10 pg of
Xwnt-8 mRNAs. (G) Vegetal blastomeres with 0.5 ng of 6-galactosidase plus 10 pg Xwnt-8 mRNAs. Figure shows slightly hyperdorsalized embryo
with bent axis.
(H and I) Injection of mRNAs into UV-ventralized stage 8 embryos with 0.5 ng of 5-galactosidase plus 10 pg of Xwnt8 mRNAs into marginal (H)
or vegetal (I) blastomeres.