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The Spemann organizer has long been recognized as a major source of patterning signals during the gastrula stage of amphibian embryogenesis. More recent evidence has suggested that the ventral side of the embryo also plays an important role in dorsal-ventral patterning during gastrulation through the action of signaling factors such as BMP-4. Bmp-4 is closely related to the Drosophila decapentaplegic (dpp) gene, and like Bmp-4, dpp is excluded from the neurogenic region. Recently we showed that Bmp-4 functions in an analogous role to that of dpp in Drosophila, suggesting that the mechanism of dorsal-ventral patterning in Xenopus and Drosophila embryos may be conserved. To further test this hypothesis, RNA of the Drosophila short gastrulation (sog) gene was injected into Xenopus embryos, since sog has been shown genetically to be an antagonist of dpp function. Overexpression of sog RNA in Xenopus dorsalizes the embryo by expanding neurogenic and dorsal paraxial tissue. When ectopically expressed on the ventral side of the embryo, sog induces a partial secondary axis. In addition, sog partially rescues embryos ventralized by ultraviolet irradiation. Since sog induces many similar changes in gene expression to that caused by truncated BMP receptors, we suggest that sog functions in part by opposing BMP-4 signaling. The recent identification of a possible Xenopus sog homolog, chordin, in conjunction with these results supports the hypothesis that dorsal-ventral patterning mechanisms are conserved between these two species.
Fig. 1. Drosophila sog induces a secondary axis in Xenopus
embryos. The top three embryos were injected ventrally with sog
RNA. The top two embryos have secondary tails and the third
embryo’s secondary axis extends from the primary axis. The bottom
embryo is an uninjected control embryo. All embryos are at stage 38.
Fig. 2. Injection of sog RNA alters early gene expression. (A,B) Vegetal view of gsc expression in stage 10 embryos. Dorsal is up.
(A) Uninjected embryo. (B) Embryo injected ventrally with 6 ng sog RNA. Gsc expression is only weakly enhanced on the ventral side.
(C,D) Vegetal view of Xnot expression in stage 11 embryos. Dorsal is up. (C) Uninjected embryo. (D) Embryo injected ventrally with 6 ng sog
RNA. Xnot expression is enhanced on the ventral side, however only in the non-notochordal domain of expression. (E,F) MyoD expression in
mid-neurula-stage embryos. Anterior is up. (E) Uninjected embryo, dorsal view. (F) Embryo injected ventrally with 4 ng sog RNA, dorsolateral
view. An additional domain of MyoD expression extends from the primary paraxial domain of expression (arrowhead). No midline clearing is
present in this secondary axial expression. (G,H) Xwnt-8 expression in mid-neurula-stage embryos. Dorsal is up. (G) Uninjected embryo,
dorsoposterior view. Xwnt-8 expression is found ventrally and laterally, and adjacent to the closed blastopore. (H) Embryos injected ventrally
with 6 ng sog RNA, posterior view. VentralXwnt-8 expression has been eliminated and the lateral expression has been extended along the sides
of the new secondary axis (2°). The primary axis is up (1°).
Fig. 3. Secondary axes induced
by sog RNA lack notochords.
(A) Posterior view of Xbra
expression in an uninjected
mid-neurula-stage embryo.
Xbra is expressed both
posteriorly, surrounding the
closed blastopore, and in the
presumptive notochord. Dorsal
is up. (B) Posterior view of a
mid-neurula-stage embryo
injected ventrally with 6 ng
sog RNA. Posterior Xbra
expression remains (although
slightly altered) and the
notochord expression along the
primary axis (1°) is present. No
axial expression of Xbra is
seen in the secondary axis (2°).
Dorsal is up. (C) Transverse
section near the tail of a stage
38 embryo (Fig. 1, third
embryo from the top). The vacuolated notochord (no) is visible in the primary axis, as are the somites (s) and neural tube (ne). The secondary
axis (to the lower right) has no notochord, but instead contains neural tissue (ne) as well as somitic tissue (s) that is fused across the midline of
the secondary axis, and with the somitic tissue on one side of the primary axis (arrowhead). (D) Transverse section of a stage 38 embryo in
which the secondary axis is opposite and distinct from the primary axis (embryo similar to Fig. 1, top embryo). The secondary axis is on the
ventral side of the embryo (bottom of picture), and does not contain a notochord. Labeling as in C.
Fig. 4. sog partially rescues uv-irradiated embryos. (A) DAI scores obtained in uninjected
(top), sog RNA-injected (middle), and ea-sog-injected (bottom) embryos. Each chart
shows the range of values between completely ventralized (DAI=0) and normal (DAI=5)
embryos, and the percentage of embryos with each DAI value. (B-D) Stage 34 embryos,
anterior is to the left. (B) Uninjected embryo. (C). Uv-irradiated embryo injected with 4 ng
wild-type sog RNA (DAI=2). (D) Uv-irradiated embryo injected with 4ng ea-sog
(DAI=2). (E) Uninjected, uv-irradiated embryo (DAI=0). Closed blastopore is to the right.
Fig. 5. Gene expression in uv-irradiated
embryos rescued by sog RNA injection.
(A-C) MyoD expression in early
neurula-stage embryos. (A) Uninjected
embryo. (B) Uv-irradiated embryo
injected with sog RNA. MyoD is
expressed in the induced axis. There is
no midline clearing of expression.
(C) Uninjected, uv-irradiated embryo.
(D-F) Xbra expression in mid-neurulastage
embryos. (D) Uninjected embryo.
(E) Uv-irradiated embryo injected with
sog RNA. Posterior Xbra expression is
present, however no notochordal Xbra is
present in the induced axis.
(F) Uninjected, uv-irradiated embryo.
Xbra is expressed around the blastopore,
but there is no axial Xbra expression.
(G,H) Hairy II expression in midneurula-
stage embryos. (G) Uninjected
embryo. (H) Uv-irradiated embryo
injected with sog RNA. Hairy II
expression is found in a stripe around
the neural plate, and demonstrates a
reduction in the size of the anterior
neural region. No dorsal midline
expression of Hairy II is observed.
(I) Uninjected, uv-irradiated embryo. In
the above panels, the uninjected and sog
RNA-injected embryos are shown in a
dorsal view with anterior up, whereas
the uninjected, uv-irradiated embryos
are shown in a posterior view. (J) Krox-
20 expression in a stage 27 control
embryo (top embryo; black arrowhead
marks the two stripes of expression in
rhombomeres 3 and 5) and in a uvirradiated
embryo injected with sog
RNA (bottom embryo). In this embryo,
both stripes of Krox-20 expression were
observed (white arrowhead). (K)
Cement gland was present in some uvirradiated
embryos injected with sog
RNA (bottom embryo, black
arrowhead). In this embryo, only a
single stripe of Krox-20 expression was
observed.
Fig. 6. uvirradiated,
soginjected
embryos do not
contain a
notochord.
Transverse
section through
a stage 37/38
uv-irradiated
embryo
injected with
sog RNA at the
8-cell stage.
The somites (s)
are fused across
the dorsal
midline beneath
the neural
tissue (ne).
Fig. 7. Ubiquitous expression of sog dorsalizes the embryo.
(A,B) Lateral view of MyoD expression at the mid-neurula stage in
(A) an uninjected embryo and (B) an embryo injected with sog RNA
in each blastomere at the 4-cell stage. Anterior is to the left. MyoD
expression extends around the entire ventroposterior region of the
injected embryo. (C,D) Transverse section of an embryo similar to
the one in B. (C) Anterior section. MyoD is expressed in the paraxial
mesoderm flanking the
presumptive notochord (no).
(D) Posterior section. MyoD is
expressed circumferentially,
except in the dorsal midline,
the region of the presumptive
notochord (no). (E) Dorsal
view of late neurula-stage
embryos. Top embryo is an
uninjected control embryo.
Bottom three embryos were
injected in each blastomere at
the 4-cell stage with ea-sog
RNA (identical results were
observed with wild-type sog
RNA). The posterior region of
the embryo was considerably
elongated and narrowed.
Anterior is to the left.
(F-H) Hairy II expression at
the late neurula stage in an
uninjected embryo (F) and in
an embryo injected with sog
RNA in each blastomere at the
4-cell stage (G,H). Dorsal view
in F, lateral view in G, and
ventral view in H; anterior is to
the left in all three panels. The
stripe of Hairy II expression
bordering the neural plate in
the injected embryo continues
around to the ventral side
(arrowhead). (I) Transverse
section of a sog-injected
embryo stained for HairyII as in G and H, cut at the
level of the stripe that circumscribes the embryo.
HairyII expression was found to continue around the
ventral side of the embryo in the ectodermal layer of
cells. Expression of Hairy II in the floorplate was
retained (arrowhead). (J) Top: Control stage 38 embryo;
bottom: stage 38 embryo injected with sog RNA (third
embryo from the top in E). A normal ventral tail fin has
not formed.
Fig. 8. BMP-4 prevents the axis-inducing capabilities of sog in uvirradiated
embryos. Top: uninjected embryo; middle: uv-irradiated
embryo injected with 5 ng sog RNA (DAI=2); bottom: uv-irradiated
embryo co-injected with 5 ng sog RNA and 300 pg Bmp-4 RNA
(DAI=0). Uninjected, uv-irradiated embryos were similar to the
bottom injected embryo and had DAI values of 0 (data not shown).
All embryos are at the equivalent of stage 37.
