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

项目总结 具有运动纤毛的适当分化的纤毛上皮的生成和维持功能障碍导致 许多人类纤毛疾病可能导致脑积水、先天性心脏缺陷、呼吸道感染、 反位和/或不孕症。鉴于参与装配、结构和运动的部件 纤毛的运动性已被鉴定，但对纤毛发生的转录控制知之甚少。在这里我们 试图通过研究纤毛上皮形成的转录调节来填补这一空白 以斑马鱼胚胎的库普弗氏囊泡(KV)为模型系统。这个有纤毛的器官起着鱼的作用 在原肠形成后不久，从其前体细胞发育而来的背侧先驱细胞 (DFC)。 我们最近发现了转录因子和辅助因子，这些转录因子和辅因子是已知的调节或调节 河马信号通路的转录结果可能是DFCs分化的主要调节因子。我们 发现它们作用于主要信号通路和已知的控制关键转录因子的上游 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，我们发现河马转录因子和辅助因子控制节点信号的活性 在DFCs分化为KV的过程中，这一途径的作用是未知的。 我们提出了三个目的来阐明河马转录因子和辅助因子的机制 控制KV纤毛上皮的形成。首先，我们将描述转录网络的特征 这直接和间接地受到这些因素的影响。然后我们将对 筛选直接靶基因以鉴定纤毛上皮形成所需的新功能 KV。其次，我们将探索河马转录因子和辅助因子之间的功能关系，通过 救援实验和分析他们的物理相互作用。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，以前在癌细胞培养系统中建立，在其中 他们的行为被认为是敌对的。第三，我们将研究河马转录的机制 辅因子控制结节的活性，例如通过DNA甲基化标记编写者的转录控制。 这些研究的完成将极大地促进我们对纤毛虫感染机制的了解 上皮细胞在转录水平上被编程。此外，通过提供一种新的交互模式 在正常发育过程中河马下游介体和调节器之间的关系，并定义组织特异性 河马转录因子和辅因子对表观遗传的转录调控，我们的结果将大大 扩大我们对河马信号通路的认识。最后，对这一代人和 在胚胎发育期间保持适当分化的纤毛上皮可应用于 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.