P38 MAP kinase and Calmodulin Signalling Pathways Act in Concert to Initiate Embryonic Genome Activation in the Mouse Embryo (#100)
The maternal-to-embryonic transition of genetic control of development is essential for development. In mammals this occurs around the 2-cell to 8-cell stage and is characterised by the activation of transcription from the new embryonic genome (EGA). In the mouse definitive EGA occurs at the 2-cell stage. Two essential transcription factors, CREB and ATF1, are activated at the time of EGA. In the zygote and 2-cell embryo CREB is activated by a Paf/PI3K/Ca2+/calmodulin/CAM kinase-dependent signalling pathway (1), and nuclear localisation of ATF1 requires the actions of a P38 MAP kinase-dependent signalling pathway (2). The purpose of this study was to undertake a pharmacological analysis of the roles of these two signalling pathways in EGA.
Zygotes were collected 18 h post-hCG and cultured in medium containing calmodulin antagonist (W-7) and/or P38 antagonist (SB203580). Both antagonists alone caused dose-dependent inhibition of development at stages after the 2-cell stage of development, but combined W-7 (7 µM) and SB203580 (10 µM) treatment caused developmental arrest at the 2-cell stage. Immunostaining showed this combined treatment caused reduced expression, activation and nuclear accumulation of both ATF1 and CREB in 2-cell embryos. Quantitative real-time RT-PCR showed that treatment with W7 + SB203580 reduced transcription of Hsp70a1b (a hallmark transcription product of EGA) and reduced overall levels of transcription, as assessed by BrUTP incorporation. This treatment also reduced progression of cells through the S-phase as assessed by BrdU incorporation.
The study showed that P38 and calmodulin act in concert to regulate CREB/ATF1 activation at the time of EGA. It shows that blocking the calmodulin and P38 MAP kinase signalling pathways prevented the normal onset of EGA and prevented normal cell-cycle progression. These findings identify new players and interactions required for the normal onset of EGA in the mouse embryo.
- Jin XL, O'Neill C. Biol. Reprod. 2011; 84: 1216-24.
- Jin XL, O'Neill C. 42nd SRB, 2011, Cairns, Australia.