The translational program in oocyte and early embryo development

描述（由申请人提供）：卵母细胞具有通过母体mRNA积累指导胚胎发育的显著特性。在没有转录的情况下，所有与受精、雄性和雌性原核的重编程以及最初几轮复制相关的关键事件都受到mRNA翻译程序的调控。使用全基因组的多核糖体分离和微阵列分析的方法，我们已经产生了在卵母细胞/合子过渡的翻译模式的蓝图。通过挖掘这些数据集以及已发表的数据，出现了卵母细胞在卵母细胞成熟早期合成受精卵中使用的机器。在高度调节的转录物中，有那些编码RNA结合蛋白Dazl和Pumilio 2的转录物，它们对无脊椎动物的生殖系发育和多能性的维持至关重要。所测试的假设是，这些RNA调节剂在早期胚胎发育过程中起着关键作用，并且作为延伸，在胚胎干细胞的多能状态中起着关键作用。提出了三个具体目标来检验这一假设。具体目标1，我们建议的特点，在卵母细胞到受精卵的过渡，包括Dazl和Pumilio 2的翻译调节机制。在特定目标2中，将通过条件性切除这些基因或通过吗啉代寡核苷酸注射破坏它们的翻译来研究Dazl和Pumilio 2在胚胎发育早期阶段的功能。这些RNA结合蛋白的作用机制将通过定义其靶点来研究。具体目标3中描述的实验将检验由这些RNA结合蛋白介导的翻译由胚胎和胚胎干细胞共享的假设。将研究这些RNA调节因子和mRNA靶点网络缺陷的ES细胞表型。该项目意义重大，因为它将揭示广泛适用于不同开发系统的法规。了解植入前胚胎发育的调控过程将揭示表观遗传调控和重编程的新范式，这将改善目前从体细胞诱导多能干细胞的策略。 公共卫生相关性：相关性拟议的研究与改善人类健康和NIH的使命有关，因为它提供了对胚胎发育机制的更好的了解，因此，它将改善遗传疾病的诊断和治疗。它还将提供新的工具来操纵生物化学机制，这些机制对于指导细胞分化以实现组织再生至关重要。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Relevance 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.