The oocyte has the remarkable property of directing the embryo development through accumulation of maternal mRNAs. In the absence of transcription, all the critical events associated with fertilization, reprogramming of the male and female pronucleus, and initial rounds of replication are regulated by a program of mRNA translation. Using a genome wide approach of polysome fractionation and microarray analysis, we have generated a blueprint of the pattern of translation at the oocyte/zygote transition. By mining these data sets as well as published data, it is emerging that the oocyte synthesizes the machinery utilized in the zygote early during oocyte maturation. Among the highly regulated transcripts are those coding for RNA binding proteins Dazl and Pumilio2, critical for germ line development and maintenance of pluripotency in invertebrates. The hypothesis tested is that these RNA regulators play a critical function during early embryo development and, as an extension, in the pluripotent state of the embryonic stem cells.
Three Specific Aims are proposed to test this hypothesis.
With Specific Aim 1, we propose to characterize the mechanism regulating translation at the oocyte to zygote transition, including the Dazl and Pumilio2.
In Specific Aim 2 the function of Dazl and Pumilio2 during the early stages of embryo development will be investigated by conditional ablation of these genes or by disrupting their translation by morpholino oligonucleotide injection. The mechanisms of action of these RNA binding proteins will be investigated by defining their targets. Experiments described in Specific Aim 3 will test the hypothesis that translation mediated by these RNA binding proteins is shared by the embryo and by embryonic stem cells. The phenotype of ES cells deficient in these RNA regulators and the network of mRNA targets will be investigated. This project is significant because it will uncover regulations that are of broad application to different developmental systems. Understanding the regulatory processes of preimplantation embryonic development will reveal new paradigms in epigenetic regulation and reprogramming that will improve current strategies of induction of pluripotent stem cells from somatic cells.
The proposed research is relevant to improving human health and the mission of NIH because it provides a better insight into the mechanisms of embryo development and, therefore, it will improve diagnosis and cure of genetic diseases. It will also provide new tools be manipulated biochemical machineries essential to direct cell differentiation for tissue regeneration.
|Lin, Chih-Jen; Koh, Fong Ming; Wong, Priscilla et al. (2014) Hira-mediated H3.3 incorporation is required for DNA replication and ribosomal RNA transcription in the mouse zygote. Dev Cell 30:268-79|