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The Xenopus Wnt-8 gene is transiently expressed in ventral and lateral mesoderm during gastrulation and plays a critical role in patterning these tissues. In the current study, we show that the spatial and temporal pattern of expression of endogenous Xwnt-8 is regulated, in part, at a post-transcriptional level. We have identified a novel sequence element in the 3' untranslated region of the Xwnt-8 RNA that controls the polyadenylation status of reporter and endogenous Xwnt-8 RNAs, directs rapid RNA degradation beginning precisely at the early gastrula stage, and represses translation of transcripts throughout development. Expression of endogenous Xwnt-8 is normally downregulated within lateral (presomitic) mesoderm following gastrulation. We demonstrate that rapid degradation of Xwnt-8 transcripts, mediated by these regulatory elements in the 3' untranslated region, is essential to this process and that downregulation is required to prevent overcommitment of somitic cells to a myogenic fate. These studies demonstrate a role for post-transcriptional regulation of zygotic gene expression in vertebrate embryonic patterning.
Fig. 1. Differential secondary axis induction and inhibition of anterior development by
injected Xwnt-8 RNAs that contain or lack 3¢ untranslated sequence. Xwnt-8 RNAs that
either include (X8/UTR+; A,C) or lack (X8/UTR-; B,D) 3¢ untranslated sequence were
injected into the dorsal (DMZ) or ventral marginal zone (VMZ) of 4-cell embryos, which
were cultured to the tadpole stage and photographed. Arrow in B indicates partial
secondary axis.
Fig. 2. Xwnt-8 RNAs that lack the 3¢ UTR can posteriorize neural
tissue and induce neural crest whereas RNAs that include the 3¢ UTR
cannot. (A) Animal caps were isolated from control (uninjected)
embryos or from embryos injected with noggin (Nog) RNA alone, or
in combination with X8/UTR+ or X8/UTR- RNAs. Ectodermal
explants were cultured to stage 25 and expression of pan-neural
(NCAM), anterior neural (OtxA), posterior neural (Xlhbox6), neural
crest (Xslug), dorsal mesodermal (muscle actin) and ubiquitously
expressed (EF1-a) genes were analyzed by RT-PCR. (B) X8/UTR+
(left panel) or X8/UTR- (right panel) RNA was injected into the
animal pole of one blastomere of 2-cell embryos and expression of
the neural crest marker Xtwist was analyzed by in situ hybridization
at stage 18. Arrow indicates expanded domain of Xtwist staining.
Fig. 3. The 3¢ UTR of Xwnt-8 induces the degradation of a
heterologous transcript beginning shortly after the onset of
gastrulation. Vim/UTR+ or Vim/UTR- RNA was injected into 1-cell
embryos. RNA was extracted from 10 embryos in each experimental
group and from uninjected (control) embryos at the indicated
developmental stages. A northern blot containing 15 mg of each RNA
was hybridized with a radiolabeled Vim antisense probe. The Vim
probe detects endogenous vimentin RNA and thus signal is observed
in control lanes by the late gastrula stage.
Fig. 4. Xwnt-8 3¢ UTR-mediated destabilization of transcripts
requires de novo protein synthesis. Vim/UTR+ or Vim/UTR-
transcripts were injected into 1-cell embryos, which were then
cultured in the presence or absence of cycloheximide (CHX) as
indicated (CHX:-, absence of CHX; 9, incubated in CHX beginning
at stage 9; 10, incubated in CHX beginning at stage 10). RNA was
extracted from 10 embryos in each experimental group (RNA: con,
uninjected controls; UTR+, Vim/UTR+ injected; UTR-, Vim/UTR-
injected) at the indicated developmental stages and the persistence of
injected RNA was assayed by northern blot hybridization using a
Vim antisense riboprobe (VIM). The same filter was rehybridized
with an antisense Xwnt-8 riboprobe (Xwnt-8).
Fig. 5. The 3¢ UTR of Xwnt-8 inhibits translation of a reporter
transcript in developing embryos. (A) Vim/UTR+ or Vim/UTR-
RNAs were injected near the dorsal side of cleaving embryos and
synthesis of Vim protein was analyzed by immunostaining whole
embryos at the indicated stages with antiserum specific for the mycepitope
tag present in the Vim reporter protein. Arrows indicate
specific staining. (B) Vim/UTR+ or Vim/UTR- RNAs were injected
into 1-cell embryos and total proteins were extracted from injected
and uninjected (control) embryos at the indicated stages. A western
blot containing three embryo equivalents of each extract was probed
with anti-myc antiserum and was reprobed with an antibody specific
for spectrin to demonstrate the presence of fairly equivalent amounts
of protein in each lane.
Fig. 6. Deletion analysis of the Xwnt-8 3¢ UTR identifies a 194 nucleotide
regulatory region. (A) Nucleotide sequence of the Xwnt-8 3¢ UTR with
deletion mutant forms indicated. UTR consists of sequence between
asterisks, sequence contained in UTR1 is underlined and UTR2 consists of
sequence between the two arrows. (B) Reporter RNAs used to test the
function of the three deletion mutants indicated in A are shown
schematically. Black box represents myc coding sequence; black line, Xwnt-
8 3¢ UTR; open box, SV40 polyadenylation signal. Reporter RNAs were
injected into 1-cell Xenopus embryos and postgastrula stage stability and
midblastula stage translation were assayed by northern analysis and wholemount
immunostaining as described in the legends to Figs 3 and 5.
Fig. 7. Endogenous Xwnt-8 RNA is inefficiently translated. (A) RNA
from stage 11 embryos was separated into polysomal (pellet) and
non-polysomal (supernatant) fractions. As a control, some samples
were adjusted to 20 mM EDTA, which dissociates RNA from
ribosomes. The presence of Xwnt-8 transcripts in each fraction was
analyzed by northern blot hybridization. (B) Northern blot analysis
of RNA extracted from MT/UTR2-injected embryos at stage 9 and
separated into polysomal and non-polysomal fractions.
Fig. 8. RNAs bearing the Xwnt-8 3¢ UTR have a short poly(A) tail.
(A) RT-PCR assay for poly(A) tail length. RNA was reverse
transcribed using an oligo(dT)/adapter primer and the resultant
cDNA amplified using primers that anneal to the 3¢ adapter sequence
and sequence in the Xwnt-8 3¢ UTR. Specific products were detected
by hybridizing Southern blots with a radiolabeled Xwnt-8 UTR
probe. (B) MT-UTR1 or MT-UTR2 transcripts were injected into
Xenopus embryos and the poly(A) tail length of each transcript was
assayed by RT-PCR amplification of RNAs extracted from injected
embryos at stage 9 in the presence (+) or absence (-) of reverse
transcriptase (RT). (C) RT-PCR analysis of poly(A) tail length of
endogenous Xwnt-8 transcripts at the indicated developmental
stages.
Fig. 9. UTR-mediated degradation of endogenous Xwnt-8 transcripts
is required for normal myogenesis. (A) Synthetic RNAs
complementary to UTR2 (A¢) or UTR1 (B¢,C¢) were co-injected with
b-galactosidase RNA into the dorsolateral marginal zone on one side
of 4-cell embryos as illustrated. This injection targets transcripts to
regions of the embryo fated to form somites (hatched) on one side of
the body. Embryos were stained for b-galactosidase activity (light
blue stain) and then hybridized with digoxigenin-labeled Xwnt-8
probe (purple stain). Staining of Xwnt-8 positive cells in the central
nervous system (arrowheads) and dorsolateral regions (arrows) is
indicated. (B) Embryos were injected with antisense UTR2 (left
panel) or UTR1 (right panel) RNAs as illustrated in A, cultured to
the tailbud stage, stained for b-galactosidase (blue) and then
immunostained with a muscle specific antibody (brown stain).
Paraffin sections of stained embryos are shown. Blocks of muscle on
the injected (arrow) and uninjected (arrowhead) side of each embryo
are indicated
