Transcriptional Control of the Formation of a Ciliated Epithelium by Downstream Mediators and Regulators of the Hippo Signaling Pathway

项目摘要 产生和维持具有运动纤毛的适当分化的纤毛上皮的功能障碍导致 许多人类纤毛病可导致脑积水，先天性心脏缺陷，呼吸道感染， 内脏逆位和/或不育。然而，组件涉及的组装，结构和运动的 纤毛的运动已经被确定，很少有人知道纤毛发生的转录调控。这里我们 试图通过研究纤毛上皮形成的转录调控来填补这一空白， 斑马鱼胚胎的Kupffer囊泡（KV）作为模型系统。这个纤毛器官的功能就像鱼 左右的组织者和发展，在原肠胚形成后不久，从它的前体，背先行细胞 （DFC）。 我们最近鉴定了已知介导或调节转录因子和辅因子， Hippo信号通路的转录产物是DFC分化的主要调节因子。我们 发现它们作用于主要信号通路的上游和已知控制转录的关键转录因子。 KV的形成，以及它们在DFC中调节表观遗传修饰酶的表达。 特别是，我们发现Hippo转录因子和辅因子控制Nodal信号传导的活性， 在DFC分化为KV的过程中，这是该途径以前未知的作用。 我们提出了三个目的来阐明Hippo转录因子和辅因子在转录过程中的作用机制。 控制KV纤毛上皮的形成。首先，我们将描述转录网络 直接或间接地受到这些因素的影响。然后，我们将执行功能分析， 选择的直接靶基因，以确定新的功能所需的纤毛上皮细胞的形成， KV其次，我们将通过以下途径探索Hippo转录因子和辅因子之间的功能关系： 救援实验和分析它们的物理相互作用。我们将挑战他们目前的模式， 在正常发育过程中的相互作用，先前在癌细胞培养系统中建立，并且其中 它们被认为是对抗性的。第三，我们将研究Hippo转录的机制， 辅因子控制Nodal活性，例如通过转录控制DNA甲基化标记的书写者。 这些研究的完成将极大地推进我们对纤毛类动物的机制的认识， 上皮细胞在转录水平上被编程。此外，通过提供新的互动模式， 在正常发育过程中Hippo下游介质和调节剂之间的关系，并定义组织特异性 Hippo转录因子和辅因子对表观遗传学的转录调控，我们的结果将大大 扩大我们对Hippo信号通路的了解。最后，对这一代人的了解， 在胚胎发育过程中维持适当分化的纤毛上皮可用于 人类纤毛疾病急需的治疗方法的发展。

Project summary Dysfunction in generating and maintaining properly differentiated ciliated epithelia with motile cilia results in a number of human ciliopathies that can lead to hydrocephalus, congenital heart defects, respiratory infections, situs inversus and/or infertility. Whereas components involved in the assembly, structure and movement of motile of cilia have been identified, little is known about the transcriptional control of ciliogenesis. Here we seek to fill this gap by investigating the transcriptional regulation of the formation of a ciliated epithelium using the Kupffer's vesicle (KV) of the zebrafish embryo as a model system. This ciliated organ functions as the fish left-right organizer and develops, soon after gastrulation, from its precursors, the Dorsal Forerunner Cells (DFCs). We recently identified the transcription factors and cofactors known to mediate or to regulate the transcriptional outcome of the Hippo signaling pathway to be master regulators of DFCs differentiation. We discovered that they act upstream of major signaling pathways and key transcription factors known to control the formation of the KV and that they regulate, in the DFCs, the expression of epigenetic modifying enzymes. In particular, we found that Hippo transcription factors and cofactors control the activity of Nodal signaling during differentiation of the DFCs into KV, a previously unknown role for this pathway. We propose three aims to elucidate the mechanisms by which Hippo transcription factors and cofactors control the formation of the ciliated epithelium of the KV. First, we will characterize the transcriptional network that is directly and indirectly regulated by each of these factors. We will then perform functional analyzes on selected direct target genes to identify novel functions required for the formation of the ciliated epithelium of the KV. Second, we will explore the functional relationships between Hippo transcription factors and cofactors via rescue experiments and analysis of their physical interactions. We will challenge the current model of their interaction during normal development, previously established in a cancer cell culture system, and in which they are thought to act antagonistically. Third, we will investigate the mechanisms by which Hippo transcription cofactors control Nodal activity, for example by a transcriptional control of writers of DNA methylation marks. The completion of these studies will greatly advance our knowledge of the mechanisms by which a ciliated epithelium is programmed at the transcriptional level. In addition, by providing a new model of interaction between Hippo downstream mediators and regulators during normal development, and defining tissue-specific transcriptional control of epigenetics by Hippo transcription factors and cofactors, our results will greatly expand our knowledge of Hippo signaling pathway. Finally, insights gained about the generation and maintenance of a properly differentiated ciliated epithelium during embryonic development can be applied to the development of much-needed treatment for human ciliopathies.