Differential Response to the Dpp Morphogen

描述(申请人提供)：在所有动物中，胚胎发生的初始事件由母体基因产物控制，这些产物沉积到发育中的卵母细胞中。在受精后的某个时间点，胚胎发生的控制权被转移到合子基因组，这一过程被称为母体到合子的转变(MZT)。在此期间，母体RNA被降解，合子RNA被转录。虽然关于RNA降解的机制已经取得了进展(Giraldez等人，2006年)，但合子基因组的激活剂仍然难以捉摸。一个线索来自于许多果蝇受精卵基因共享与CAGGTAG相关的顺式调控基序(ten Bosch等人，2006年；De Renzis等人，2007年)。我们最近分离出一种锌指蛋白，Zelda，它专门与这些位点结合。缺乏母体Zelda转录本的突变胚胎不能激活受精卵基因样本的转录，并且在细胞化过程的几个方面存在缺陷，这对应于胚胎不能再仅依靠母体产品存活的点(Merrill等人，1988年)。我们的初步结果表明，塞尔达在MZT过程中激活了一系列基因，每个基因都负责一个关键的发育过程。Zelda可能会激活介导母体RNA降解的早期转录的microRNAs，从而在MZT的两个标志性事件之间提供联系。在我们初步数据的基础上，我们将进一步描述塞尔达在MZT中的作用。塞尔达激活了多少基因，哪些早期基因是独立于塞尔达的？我们将在目标1中使用基因组学方法来确定早期胚胎中的所有塞尔达靶点。塞尔达激活了多少基因，是否有几组基因不受塞尔达的调控？在目标2中，我们询问Zelda在激活目标基因方面是起指导作用还是允许作用，或者两者兼而有之，以及它是如何与其他关键调控因子如背部和DPP相互作用来激活它们的目标基因的。在目标3中，我们研究了保守的Zelda蛋白结构域的结构和功能，并测试在长胚种Nasonia黄蜂中是否存在Zelda功能，以及它是否在激活合子基因组方面起到类似的作用。最后，我们解决了与AIM 4中基因组激活的时机有关的机械性问题，以及塞尔达在逆转基因组最初沉默方面的可能作用。这项提案中概述的实验有可能极大地促进我们对MZT潜在机制的理解。 公共卫生相关性：研究基本发育过程背后的分子机制与理解人类疾病(如癌症)和人类疾病(如出生缺陷)的原因和进展有关。

DESCRIPTION (provided by applicant): In all animals the initial events of embryogenesis are controlled by maternal gene products that are deposited into the developing oocyte. At some point after fertilization, control of embryogenesis is transferred to the zygotic genome in a process called the maternal to zygotic transition (MZT). During this time maternal RNAs are degraded and zygotic RNAs are transcribed. Although progress has been made on the mechanisms underlying RNA degradation (Giraldez et al., 2006), the activators of the zygotic genome have remained elusive. A hint came from the discovery that many Drosophila zygotic genes share a cis-regulatory motif related to CAGGTAG (ten Bosch et al.,2006; De Renzis et al., 2007). We recently isolated a zinc-finger protein, Zelda, which binds specifically to these sites. Mutant embryos lacking maternal zelda transcripts fail to activate the transcription of a sample of zygotic genes, and are defective in several aspects of the cellularization process, which corresponds to the point at which embryos can no longer survive on maternal products alone (Merrill et al., 1988). Our preliminary results suggest that Zelda activates batteries of genes during the MZT, each responsible for a key developmental process. Zelda may activate early-transcribed microRNAs that mediate maternal RNA degradation, thus providing a link between the two hallmark events of the MZT. Building on our preliminary data, we will further characterize the role of Zelda in the MZT. How many genes does Zelda activate and which early genes are independent of Zelda? We will use a genomics approach in Aim 1 to determine all Zelda targets in the early embryo. How many genes does Zelda activate, and are there groups of genes not regulated by Zelda? In Aim 2 we ask whether Zelda plays an instructive or permissive role, or both, in activating target genes, and how it interacts with other key regulators such as Dorsal and Dpp to activate their target genes. In Aim 3 we investigate the structure and function of conserved Zelda protein domains, and test whether there is Zelda function in the long germ Nasonia wasp and if it plays a similar role in activating the zygotic genome. Finally, we address mechanistic questions regarding the timing of genome activation in Aim 4, and the possible role of Zelda in reversing the initial silencing of the genome. The experiments outlined in this proposal have the potential to advance greatly our understanding of the mechanisms underlying the MZT. PUBLIC HEALTH RELEVANCE: The study of the molecular mechanisms underlying basic developmental processes is relevant to understanding the cause and progression of human diseases such as cancer, and human disorders such as birth defects.