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The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage
N Yamauchi1, A A Kiessling, G M Cooper
1Division of Molecular Genetics, Dana-Farber Cancer Institute, Boston, MA 02115.
Abstract:
We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.
Insights
The Ras/Raf signaling pathway is crucial for mouse embryo development at the two-cell stage. Inhibiting Ras function blocks early embryo growth, but this effect is specific to this critical developmental transition.
Area of Science:
- Developmental Biology
- Cell Signaling
- Molecular Embryology
Background:
- The Ras signaling pathway plays a vital role in cellular processes, including proliferation and differentiation.
- Understanding the specific roles of signaling pathways during early embryonic development is essential for comprehending developmental mechanisms.
Purpose of the Study:
- To investigate the role of the Ras/Raf signaling pathway in mouse oocyte maturation and early embryonic development.
- To determine the specific stage at which Ras function is required during mouse embryogenesis.
Main Methods:
- Microinjection of antisense oligonucleotides, monoclonal antibodies (anti-Ras Y13-259), and dominant-negative Ras N-17 mutants into mouse oocytes and embryos.
- Assessment of meiotic progression, fertilization, and embryonic development up to the blastocyst stage.
- Rescue experiments involving co-injection with an active raf oncogene.
Main Results:
- Interference with Ras expression or function using antisense oligonucleotides or anti-Ras antibodies did not impede oocyte meiosis.
- Microinjection of Ras N-17 into fertilized eggs inhibited development to the two-cell stage, an effect rescued by co-expressing active raf.
- Microinjection of Ras N-17 into late two-cell embryos did not affect subsequent development, indicating a specific requirement at the two-cell transition.
Conclusions:
- The Ras/Raf signaling pathway is specifically required for mouse embryo development at the two-cell stage.
- This requirement likely relates to the transition from maternal to embryonic gene expression.
- Autocrine growth factor stimulation may activate the Ras/Raf pathway at this critical developmental window.