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Published on: February 2, 2016
Antisense inhibition of Xbrachyury impairs mesoderm formation in Xenopus embryos
Natalia Giovannini1, Duri Rungger
1Station de Zoologie Expérimentale, Department of Animal Biology, University of Geneva, CH-1224 Chêne Bougeries, Switzerland.
Abstract:
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.
Insights
Inhibiting Xbrachyury (Xbra) gene expression in Xenopus embryos disrupted mesoderm formation and axis development. Restoring Xbra levels partially normalized gene expression, highlighting Xbra's crucial role.
Area of Science:
- Developmental biology
- Molecular genetics
- Xenopus laevis research
Background:
- The Xbrachyury (Xbra) gene is critical for mesoderm formation and axis patterning in vertebrate embryos.
- Understanding Xbra's precise function requires precise genetic manipulation techniques.
Purpose of the Study:
- To investigate the functional role of the Xbrachyury (Xbra) gene in Xenopus laevis development using antisense RNA inhibition.
- To analyze the downstream effects of Xbra gene inhibition on mesodermal gene expression and embryonic axis formation.
Main Methods:
- In situ synthesis of antisense RNA targeting Xbra mRNA using an RNA polymerase III expression vector.
- Injection of antisense RNA into fertilized eggs or 2-cell stage Xenopus embryos.
- Quantification of Xbra mRNA and protein levels, and analysis of mesodermal gene expression (Xvent, pintallavis, Xlim, Xwnt-8, noggin, goosecoid).
Main Results:
- Antisense inhibition markedly reduced Xbra mRNA and protein levels, leading to deficiencies in mesodermal derivatives and axis formation.
- Posterior axis organization was significantly affected, with some embryos failing to form mesoderm.
- Downstream mesodermal gene expression was reduced, except for goosecoid, and could be partially normalized upon rescue by Xbra overexpression.
- Dose-dependent effects and rescue experiments confirmed the specificity of the antisense inhibition.
Conclusions:
- Antisense inhibition of Xbra is an effective method to study its developmental role in Xenopus.
- Xbra is essential for proper mesoderm formation and axis patterning, influencing the expression of numerous downstream genes.
- Comparing antisense and dominant-negative inhibition approaches is recommended for comprehensive functional gene analysis in Xenopus development.
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