The translational program in oocyte and early embryo development

描述（申请人提供）：卵母细胞具有通过母体mrna的积累来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、雌雄原核重编程和初始复制相关的关键事件都由mRNA翻译程序调节。利用多体分离和微阵列分析的全基因组方法，我们已经生成了卵母细胞/受精卵转变时翻译模式的蓝图。通过挖掘这些数据集以及已发表的数据，我们发现在卵母细胞成熟的早期，卵母细胞合成了合子中使用的机制。在高度调控的转录本中，编码RNA结合蛋白Dazl和Pumilio2的转录本对无脊椎动物生殖系的发育和多能性的维持至关重要。经过验证的假设是，这些RNA调节因子在胚胎早期发育过程中起着关键作用，并延伸到胚胎干细胞的多能状态。提出了三个具体目标来验证这一假设。在Specific Aim 1中，我们拟对包括Dazl和Pumilio2在内的卵母细胞向受精卵转变过程中调节翻译的机制进行表征。在特异性目标2中，将通过有条件地消融这些基因或通过注射morpholino寡核苷酸破坏它们的翻译来研究Dazl和Pumilio2在胚胎发育早期的功能。这些RNA结合蛋白的作用机制将通过确定它们的靶标来研究。具体目标3中描述的实验将验证由这些RNA结合蛋白介导的翻译是由胚胎和胚胎干细胞共享的假设。研究人员将研究缺乏这些RNA调节因子和mRNA靶标网络的胚胎干细胞的表型。这个项目意义重大，因为它将揭示广泛适用于不同发展体系的规则。了解胚胎着床前发育的调控过程将揭示表观遗传调控和重编程的新范式，这将改善目前体细胞诱导多能干细胞的策略。

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.