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Studies in Xenopus have shown that growth factors of the TGF beta and Wnt oncogene families can mimic aspects of dorsal axis formation. Here we directly compare the inductive properties of two Wnt proteins by injecting synthetic mRNA into developing embryos. The results show that Wnt-1 and Xwnt-8 can induce a new and complete dorsal axis and can rescue the development of axis-deficient, UV-irradiated embryos. In contrast, activin mRNA injection induces only a partial dorsal axis that lacks anterior structures. These studies demonstrate that the mechanism of Wnt-induced axis duplication results from the creation of an independent Spemann organizer. The relationship between the properties of the endogenous dorsal inducer and the effects of Wnts and activins is discussed.
Figure 1. The Effects of Injected Activin and Wnt mRNA Depend on the Site of Injection
A single prospective dorsal (A and S) or ventral (C and D) vegetal blastomere of cleavage stage embryos (6-16 cells) was injected with l-2 pg of
Xenopus activin BB mRNA (A and C) or 2-4 pg of mouse Wnt-1 mRNA (B and D). Injected embryos were allowed to develop for 2 days at room
temperature. Control embryos, injected with 5 pg of AAct RNA, were indistinguishable from normal tadpoles or from the tadpoles injected dorsally
with Wnt-1 RNA (as in [B]). Primary (lo) and secondary (29 dorsal axes are indicated. Note that the secondary axis in (A) begins at midtrunk and
extends posteriorly to form a split tail; in (C) it is also incomplete lacking anterior (head) structures, while in (D) the duplicated axis is so similar to
the primary axis that we cannot distinguish one from the other.
Figure 2. Lineage Tracing Reveals the Formation of Dorsal Structures by the Progeny of a Ventral Blastomere Injected with Wnt RNAs
A single prospective ventro-vegetal blastomere of cleavage stage (3-32 cells) embryos was injected with fluoresceinated dextran mixed with 3 pg
of mouse Wnt-1 RNA (A and B), 2 pg of Xwnt-3 RNA (C and D), or 4 pg of AAct RNA (E and F). After 2 days of development, embryos were fixed,
sectioned, and examined with darkfield (A, C, and E) and fluorescence optics (B, D, and F). (A), (B), (E), and (F) are transverse sections; (C) and
(D) are longitudinal sections. Note two notochords and four eyes in (C). but only one notochord contains the lineage tracer. Abbreviations are as
follows: n, neural tube; nc, notochord; m, muscle; e, eye. Note that the lineage tracer is found in only one of the two dorsal axes. Labeled tissues
include notochord (B and D), somites, neural tube, and some endodermal cells (B). The scale bar in (F) also applies to (A), (B), and(E) and represents
100 pm. The scale bar in (D) also applies to (C) and represents 160 pm.
Figure 3. Two Injections of Xwnt-5 RNA Produce Triple Axes
Injections were done as described in Figure 2 into two spots located equatorially at equal distances (approximately 120°) from the presumptive
dorsal midline. (A) Morphology of an injected embryo with triple axis. Note three prominent cement glands (cg), identified as dark brown formations,
secreting gluing substances. (B) Same embryo, lateral view. (C) Same embryo sectioned longitudinally. Note three notochords, one of which is
indicated
Figure 4. Xwnt, but Not Activin mRNA, Can Completely Rescue Dorsal Axis Formation in UV-Treated Axis-Deficient Embryos
Injections were done into UV-treated embryos as described in Figure 2 with AAct RNA (A), Xwnt-6 RNA (B), and activin 56 RNA (C). Normal control
tadpoles are shown in (D). Arrows in (C) point to partial axial structures observed after activin RNA injections. Arrow in (B) points to a ventral bulge
in rescued embryos, suggesting that endoderm may have some abnormalities.
Figure 5. Injection of Xwnt-8 RNA Induces New Spemann Organizer
Embryos were injected with Xwnt-8 (B, D, and E), activin be (C, F, and G), or AAct (A) mRNA into a presumptive ventral vegetal blastomere at the
8- to l&cell stage Stage 10 gastrulas (A, 6, and C) show the formation of two blastopore lips in embryos injected with Wnt and activin mRNAs.
Arrow in (C) points to a vertical invagination lip formed as a result of activin RNA injection on the presumptive ventral side. The normal dorsal lip
is on the opposite side and is not visible. Morphology of the recipient embryos that received a blastocoel graft of ventral marginal zone pieces from
embryos injected with Xwnt-8 (D and E) or with activin be (F and G) mRNAs are shown. Histological sections (E and G) of such embryos show double
head structures (E) or a secondary axis with some neural and muscle tissue formed (G). Abbreviations are the same as for Figure 2 except that
bv is brain ventricle and bpl is blastoporal lip.
Figure 6. Autonomous Differentiation of Marginal
Zone Explants from Embryos Injected
with Xwnt-6 or Activin RNA
Presumptive ventral marginal zone pieces
were explanted from embryos injected with
Xwnt-6 (D), activin (C), and AAct (A) mRNAs
at the 6- to 16cell stage into a ventro-vegetal
blastomere, and cultured for 40 hr. Control dorsal
marginal zone explants are shown in (B).
Note the prominent cement glands (cg) in (B)
and (D). The scale bar represents 400 pm.
Figure 7. Wnt Products Induce Mesoderm in Animal Cap Explants
Embryos were injected in the animal pole at the l-ceil stage with distilled
water (lane 1). with 4 pg of activin 3s mRNA (lane 2). 8 pg of Wnt-1
RNA (lane 3) or 8 pg of Xwnt-8 (lane 4). Animal caps (about the upper
one-fifth of the embryo) were cut from the injected embryos and cultured.
After 2 days RNA was extracted and probed on a Northern blot
with a mixture of “P-labeled fibronectin and muscle actin antisense
RNAs. RNA from four animal cap equivalents is in each lane.
