XB-ART-12761Development 1999 Aug 01;12615:3371-80. doi: 10.1242/dev.126.15.3371.
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Post-transcriptional regulation of Xwnt-8 expression is required for normal myogenesis during vertebrate embryonic development.
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.
PubMed ID: 10393116
Article link: Development
Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a hoxa9 hoxb9 hoxc9 myc ncam1 nog snai2 twist1 uts2r vim vim.2 wnt8a
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|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|