June 1, 1995;
Dorsalizing and neuralizing properties of Xdsh, a maternally expressed Xenopus homolog of dishevelled.
Signaling factors of the Wnt proto-oncogene family are implicated in dorsal axis formation during vertebrate development, but the molecular mechanism of this process is not known. Studies in Drosophila have indicated that the dishevelled
gene product is required for wingless (Wnt1
homolog) signal transduction. We demonstrate that injection of mRNA encoding a Xenopus homolog of dishevelled
) into prospective ventral
mesodermal cells triggers a complete dorsal axis formation in Xenopus embryos. Lineage tracing experiments show that cells derived from the injected blastomere
contribute to anterior
and dorsal structures of the induced axis. In contrast to its effect on mesoderm
, overexpression of Xdsh
mRNA in prospective ectodermal cells triggers anterior
differentiation. These studies suggest that Wnt signal transduction pathway is conserved between Drosophila and vertebrates and point to a role for maternal Xdsh
product in dorsal axis formation and in neural induction.
[+] show captions
Fig. 1. The deduced Xdsh amino acid sequence and its comparison with Drosophila dsh and mouse
dsh homolog (Dvl) proteins. The Xdsh ORF starts with the first available methionine and encodes a
putative protein of 736 residues. Proline-rich sequences are in italics. A discs large homology
region (DHR) is underlined.
Fig. 2. Xdsh transcripts are present maternally and are equally distributed in different regions of the early blastula. Total RNA isolated from embryos at different developmental stages or from embryonic explants was analyzed by Northern blotting with specific antisense RNA probes. (A) Expression of Xdsh during embryogenesis: E, fertilized eggs; B, stage 7 blastulae; G, stage 11 gastrulae; N, stage 15 neurulae and T, tailbud embryos. (B) Spatial distribution of the Xdsh transcripts in stage 7 blastulae. Explants were isolated from A, animal; M, marginal; V, vegetal; D, dorsal; V¢, ventral regions; T, RNA prepared from whole embryos. Two embryo equivalents of total RNA were loaded per each lane. FN, Xwnt8 and Vg1-specific probes were used as controls. Fibronectin RNA (FN) is a control for loading. Xwnt8 transcripts are known to appear only after the midblastula transition (Christian et al., 1991). Vg1 RNA is a vegetally localized maternal mRNA (Rebagliati et al., 1985).
Fig. 3. The effect of Xdsh mRNA depends on the site of injection. A single prospective dorsal (A) or ventral (B,D) vegetal blastomere of
cleavage-stage embryos (8-16 cells) was injected with 0.4 ng of Xdsh mRNA. Phenotypes of the injected neurulae (A,B) and of tadpoles at
stages 40-42 (C,D) are presented. Axis duplications are clearly visible in embryos injected with Xdsh RNA in a ventral blastomere (B,D).
Embryos, injected ventrally with 0.4 ng of a control ÆXdsh mRNA (C), are indistinguishable from normal tadpoles or from the tadpoles,
injected dorsally with Xdsh mRNA (as in A). Note that three embryos in D have completely duplicated body axes including most anterior and
posterior structures, whereas in one embryo both dorsal axes oppose each other and posterior development is inhibited.
Fig. 4. Histological analysis of embryos injected with Xdsh mRNA.
(A) Transverse section of an embryo injected ventrally with a control
RNA encoding b-gal; (B,C) Embryos injected with Xdsh RNA;
(B) transverse section and (C) horisontal section. Abbreviations are
as follows: nt, neural tube; nc, notochord; s, somite; e, eye; o, otic
vesicle, b, brain. The scale bar in A is 150 mm (also applies to B).
The scale bar in C represents 300 mm.
Fig. 5. Xdsh mRNA rescues dorsal development in axis-deficient UV-treated embryos.
(A) Normal control embryos. (B) Embryos ventralized by UV treatment. (C) UV-treated
embryos that were injected at the 8-cell stage in a vegetal blastomere with 0.4 ng of Xdsh
mRNA. (D) Histological analysis of ventralized embryos (top) and embryos rescued by Xdsh
mRNA injection (bottom). The scale bar in D represents 200 mm. Abbreviations are the same as
in Fig. 4, except k, kidney tubules. Note that, in C, the most anterior morphological structures
including eyes and cement glands are rescued by Xdsh RNA.
Fig. 6. Lineage tracing reveals the formation of dorsal and anterior structures by the progeny of a single blastomere injected with Xdsh mRNA.
Cleavage-stage (8-32 cells) embryos were coinjected with 0.4 ng of Xdsh mRNA and 0.2 ng of b-gal RNA or with b-gal RNA alone. After 2
days of development embryos were fixed, and stained for b-gal activity. (A) Normal embryos were injected at the 8- to 16-cell stage into a
ventrovegetal blastomere with b-gal RNA (two embryos on the top) or with b-gal and Xdsh mRNAs (bottom). (B,C) UV-treated embryos
rescued by Xdsh RNA and coinjected with b-gal RNA. (B) Staining is mainly in the pharyngeal endoderm and head mesenchyme of the fully
rescued embryos. A control embryo injected with b-gal RNA only is shown at the top. (C) In the partially rescued embryos (0.05-0.1 ng of
Xdsh RNA injected), the staining is in the notochord and pharyngeal endoderm. (D-F) Lineage tracing at the 32-cell stage. Embryos were
injected into D4 (tier 4) vegetal blastomere with b-gal RNA (D) or with b-gal and Xdsh mRNAs (E). (F) Xdsh and b-gal RNAs were
microinjected into C4 (tier 3) subequatorial blastomere. Staining is mainly in the notochord and anterior mesoderm.
Fig. 7. Overexpression of Xdsh mRNA leads to
neuralization of animal cap explants. Animal caps
from embryos injected with 1 ng of Xdsh RNA
were explanted from the injected embryos at stage 8
and cultured in isolation until the equivalent of
stage 31 (A) or stage 11 (B). After culture, the
explant RNA was extracted and analyzed by
northern blotting using different 32P-labeled
antisense RNA probes. RNA from ten animal cap
equivalents or from two embryos is loaded per lane.
The same blot was stripped and reprobed with different probes. Two major specific transcripts of
NCAM are shown (Kintner and Melton, 1987). EF1a and fibronectin probes are controls for loading.
The muscle-specific actin probe weekly cross-hybridizes with cytoskeletal actin RNAs (the two bands
above muscle-specific band). Lane 1, animal caps from embryos injected with 1 ng of Xdsh mRNA;
lane 2, animal caps from embryos injected with 0.12 ng of noggin mRNA; lane 3, control animal caps
from uninjected embryos; lane 4, sibling embryos, stage 31 (A) or 11 (B).