Giovannini N and Rungger D (2002), Antisense inhibition of Xbrachyury impairs meso...

XB-ART-7349

Dev Growth Differ 2002 Apr 01;442:147-59. doi: 10.1046/j.1440-169x.2002.00630.x.

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Antisense inhibition of Xbrachyury impairs mesoderm formation in Xenopus embryos.

Giovannini N , Rungger D .

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Expression of the Xbrachyury (Xbra) gene was inhibited by antisense RNA synthesized in situ from an expression vector read by RNA polymerase III, injected into the fertilized egg or the 2-cell stage embryo of Xenopus laevis. Antisense-treated embryos had markedly reduced levels of Xbra mRNA and protein, and showed deficiencies in mesodermal derivatives and axis formation. In particular, organization of the posterior axis was affected, but often the anterior axis was also reduced. Some embryos failed to form mesoderm altogether and remained amorphous. The antisense effect is dose-dependent and may be "rescued" by overexpression of Xbra. In Xbra-deficient embryos, expression of several mesodermal genes (Xvent, pintallavis, Xlim, Xwnt-8 and noggin) was reduced to varying degrees, whereas goosecoid levels remained normal. The modified expression levels were partly normalized when Xbra deficiency was rescued. The observation that antisense inhibition yields slightly different phenotypes from dominant-negative inhibition suggests the recommendation of using several surrogate genetic approaches to determine the functional role of a gene in Xenopus development.

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Species referenced: Xenopus laevis
Genes referenced: actc1 actl6a foxa4 gsc lhx1 tbxt ventx1.2 wnt8a

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	Fig. 1. Sense and antisense vectors. (a) Map of the Xbra cDNA with location of the sequences (filled boxes) inserted in either orientation into the VAI to produce the sense or antisense vector. BRAUG spans the AUG start and BRAMID covers a region localized 150 bp further downstream. (b) VAI clone, with the adenovirus II a1 gene (open box), and the inserted oligonucleotide (filled box). The arrows indicate the orientation of the oligonucleotide and the corresponding position of the reconstituted BamHI site (b). The intragenic VAI promoter is marked by hatched boxes. Restriction sites for HindIII (H), PstI (P), HindII (Hd), XbaI (X) and EcoRI (R) are indicated.
	Fig. 2. Reduction of Xbra expression in antisense-treated embryos. (A) Whole-mount in situ hybridization with antisense Xbra RNA probe. Left, untreated control embryo (Co) showing normal Xbra expression around the blastopore. Center, embryo carrying the BRAMID– antisense vector with near total suppression of Xbra mRNA. Right, antisense-treated gastrula with only slightly reduced Xbra expression. The clearing treatment reveals the blastocoele outline (brown). (B) Embryos injected into one cell of the 2-cell stage embryo with a mixture of antisense vector and Texas Red dextran. The fluorescent marker is localized to one body half of the living gastrula (Red). After in situ hybridization for Xbra mRNA (Bra), the marker is still recognizable in the uncleared embryo (Red 2). Note the absence of Xbra mRNA in the injected body half carrying both the fluorescent marker and the antisense vector. (C) Reverse transcription (RT)–polymerase chain reaction (PCR) analysis on single gastrulae. The uninjected control (Co) and the three BRAMID+ injected (sense construct) embryos yield similar signals. The three BRAMID– antisense embryos show strongly reduced Xbra bands. EF1 expression varies little. Aliquots of cDNA from a BRAMID+ control (c) and a BRAMID– antisense (a) embryo were further analyzed for the presence of other mesodermal mRNA (Fig. 6A). (D) Whole-mount staining of BRAMID– injected embryos (stage 17, anterior pole left) with the anti TN1-123 antibody specific for brachyury protein. One embryo shows a strong reduction in Xbra protein, the other shows normal Xbra expression in the notochord and the caudal mesoderm. Both antisense embryos were processed in the same vial.
	Fig. 3. Phenotypes of embryos carrying the BRAMID– antisense vector. (A) Normal stage 38 control embryo, (B) posterior defect, (C,D) pronounced antero–posterior defect and (E) amorphous phenotype. (F) Relative frequency of the various phenotypes in uninjected (CO) and BRAMID+ (MID+), VAI vector, BRAMID– (MID–) or BRAUG– (AUG–) injected embryos. Phenotypes considered to be Xbra specific are plotted above the zero line: amorph, no or rudimentary axis; a-p, antero-posterior defect; heavy a-p, strong shortening of both axes; p, posterior defect. Non-specific alterations are plotted below this line: dupl ax, axis duplication; microc, microcephaly.
	Fig. 4. Effect of antisense treatment on morphology of somites. (A) Longitudinal section through a short-tailed, antisense-treated embryo (stage 41). The somites in the tailroot are disorganized and poorly differentiated. (B) Detail of somitic disorganization. No clear segmental organization is established. The few muscle fibrils lack clear striation and do not associate into fibers. (C) Normally organized muscle in the tail of a sibling control embryo. Bars, 50 μm.

	Fig. 6. Changes in the level of mesodermal mRNA in Xbra antisense (A) and rescued (B,C) embryos. (A) Reverse transcription (RT)–polymerase chain reaction (PCR) analysis of various mRNA on cDNA from single embryos injected with 200 pg of BRAMID+ (control, c) or BRAMID– (antisense, a) constructs (samples a and c in Fig. 2C). In three aliquots (top line) Ef1 was amplified in the same tube. (B) RT–PCR on pools of five embryos injected with a mixture of 100 pg each of BRAMID– and BRAUG+ showing strong inhibition (a), embryos carrying BRAMID– and CMV-Xbra rescue vector and overexpressing Xbra (r) or uninjected controls (c). (C) Aliquots of the a, r, and c samples in B were further tested for the levels of mRNA of Xvent-1, pintallavis (Pint), Xlim-1, Xwnt-8, and goosecoid (gsc). M, molecular size marker.