Proc Natl Acad Sci U S A
August 29, 1995;
Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos.
The dorsoventral axis is established early in Xenopus development and may involve signaling by Wnts, a family of Wnt1
-protooncogene-related proteins. The protein kinase shaggy functions in the wingless/Wnt signaling pathway, which operates during Drosophila development. To assess the role of a closely related kinase, glycogen synthase
kinase 3 beta (GSK-3
beta), in vertebrate embryogenesis, we cloned a cDNA encoding a Xenopus homolog of GSK-3
beta-specific transcripts were detected by Northern analysis in Xenopus eggs and early embryos. Microinjection of the mRNA encoding a catalytically inactive form of rat GSK-3
beta into a ventrovegetal blastomere
of eight-cell embryos caused ectopic formation of a secondary body axis containing a complete set of dorsal and anterior
structures. Furthermore, in isolated ectodermal explants, the mutant GSK-3
beta mRNA activated the expression of neural tissue
markers. Wild-type XGSK-3
beta mRNA suppressed the dorsalizing effects of both the mutated GSK-3
beta and Xenopus dishevelled
, a proposed upstream signaling component of the same pathway. These results strongly suggest that XGSK-3
beta functions to inhibit dorsoventral axis formation in the embryo
and provide evidence for conservation of the Wnt signaling pathway in Drosophila and vertebrates.
Proc Natl Acad Sci U S A
[+] show captions
References [+] :
FIG. 1. Deduced XGSK-3P3 amino acid sequence and its comparison
with the Drosophila shaggy and rat GSK-3f3 proteins.
FIG. 2. XGSK-3f transcripts are present maternally and are uniformly
distributed in the early embryo. (A) Northern analysis of
XGSK-3,3 expression throughout embryogenesis. Developmental
stages: 2, two-cell stage; 9, late blastula; 11, midgastrula; 12, late
gastrula; and 20, late neurula. (B) Spatial distribution of the XGSK-3f3
transcripts in stage 10 gastrulae. Explants were isolated from the
animal pole region (AP), the dorsal marginal zone (DM), the ventral
marginal zone (VM), and the vegetal pole region (VP). Two embryo
equivalents of RNA are loaded per lane for Northern analysis; 18S
RNA and fibronectin bands reflect loading. Antisense probes:
brachyury, general mesodermal marker; Xwnt8, ventrolateral marker;
and goosecoid, dorsal marginal zone marker. The bar on the right of
A indicates position of 28S rRNA.
FIG. 3. Effects of GSK-3,B RNAs on embryonic development.
Embryos were injected with 1 ng of K85R GSK-3,3 mRNA (A) or
wild-type GSK-3p mRNA (B) into a ventrovegetal blastomere at the
eight-cell stage. (X6.) (C) Representative transverse section of an
embryo injected with K85R mRNA; b, brain; nc, notochord; s, somite.
(Scale bar = 150 ,um.) (D) Effect of K85R RNA on early mesodermal
markers in the marginal zone. Dorsal (D) and ventral (V) halves were
dissected from normal embryos or from embryos injected with K85R
or rat GSK-3,B RNAs and subjected to Northern analysis (RNA from
four explants is in each lane).
FIG. 4. Enzymatic activities of different forms of GSK-3f3. Lysates
were prepared at late blastula stages from embryos injected with
Myc-tagged RNA constructs: lanes 1, Xenopus GSK-3,B; lanes 2, rat
GSK-3p3; lanes 3, K85R GSK-3p3; and lanes 4, no injection. (A) Immune
complex kinase assays with anti-Myc antibodies and with myelin basic
protein (MBP) as substrate. (B) Western analysis of embryonic lysates
with anti-Myc antibodies.
FIG. 5. Wild-type XGSK-3f overcomes the effect of the K85R
mutant on dorsoventral axis formation. Embryos were injected with
0.5 ng of K85R RNA in the ventrovegetal blastomere at the eight-cell
stage (A) or with a mixture of 0.5 ng of K85R RNA and 0.25 ng of the
wild-type XGSK-3,B RNA (B) and allowed to develop until stage 39.
6. Neuralization of ectodermal explants by K85R GSK-303.
Both blastomeres of the two-cell embryos were injected with different
mRNAs as indicated. Animal cap explants were isolated at stage 8 and
cultured until stage 11 (A) for early marker analysis or until stage 28
(B) for late marker analysis. Early mesodermal markers are the same
as in Fig. 2. Otx2 and XIF3 are anterior neural markers, XAG1 is a
cement gland marker, XA1 is an anterior ectoderm marker, and
NCAM is a panneural marker. Fibronectin mRNA and 28S rRNA
bands reflect loading. Animal cap RNA was derived from uninjected
embryos (lane 4) or from embryos injected with 1 ng of K85R mRNA
(lane 1), with 1 ng of Xdsh mRNA (lane 2), with 100 pg of noggin
mRNA (lane 3). Sibling embryo RNA is in lane 5. Total RNA from
10 animal caps or from 2 embryos is loaded per lane. The same blot
was stripped and reprobed with different probes.
Protein kinase C zeta isoform is critical for mitogenic signal transduction.