Deciphering the role of p63 in secondary palate development using systems biology

上颚的发育最终将口腔与鼻道分开，它涉及一系列复杂的相互依赖的事件，需要细胞生长、细胞粘附、细胞迁移、细胞分化和细胞死亡的密切协调。如果这些过程不能正确发生，就会导致先天性腭裂。腭裂的发病率约为每2500名活产婴儿中就有1名腭裂患者，这给患者带来了相当大的问题，因为他们可能会因为面部外观的改变而出现饮食、说话和心理社会适应方面的问题。腭裂的频繁发生和主要的医疗负担突出了解剖腭裂正常发育的基本机制以及这些机制如何在腭裂中受到干扰的必要性。出于伦理考虑，这些研究不能在人类身上进行。小鼠的上颚发育反映了人类的情况，因此小鼠是研究腭发育的主要动物模型。尽管小鼠上颚发育的描述性胚胎学已经建立，但潜在的遗传事件却没有得到很好的描述。最近，我们有令人信服的证据表明转录因子p63是一个主控基因，在驱动腭发育的遗传事件中起着核心作用。我们利用高通量RNA测序技术（RNA-seq），从仔细解剖的小鼠上颚中分离出来，获得了上颚发育过程中关键时间点表达基因的完整图谱。同时，我们利用ChIP-seq技术确定了发育中腭中p63结合的所有基因组区域。尽管产生了这些全基因组数据集，并且在次腭中确认了大量p63转录靶点，但p63控制的基因调控网络尚未被阐明。这些复杂网络的行为不能用简单的直观方法来预测，而是需要复杂的计算方法。在这个项目中，我们将首先应用时间过程方法来分析从p63功能被破坏的小鼠解剖的细胞和腭架中产生的RNA-seq数据。通过结合这两个不同但互补的系统的结果，我们将确定p63控制的发育调控网络中直接的和生物学相关的早期调控联系，这将有助于我们理解p63在第二腭发育中的作用。其次，我们将使用由p63控制的基因调控网络产生的数据来分析由于p63信号增加而导致腭裂的小鼠。这些实验将使我们能够确定为什么在腭正常发育过程中，p63信号水平在特定细胞亚群中降低是必不可少的。最后，为了促进协同工作，我们将确保项目中产生的所有数据都可以通过万维网免费获得。最终，该项目将提供具有重要意义的发育过程的关键信息，并作为一个测试案例，使用系统级发育遗传学来更广泛地剖析在先天性人类畸形中被破坏的基因调控网络。

Development of the palate, which ultimately separates the mouth from the nasal passages, involves a complex series of inter-dependent events that requires close co-ordination of cell growth, cell adhesion, cell migration, cellular differentiation, and cell death. Failure of these processes to occur correctly results in the birth defect cleft palate. Cleft palate, which has an incidence of approximately 1 in 2500 live births, results in considerable problems to affected individuals as they may experience problems with eating, speaking and psychosocial adjustment due to the altered facial appearance. The frequent occurrence and major healthcare burden imposed by cleft palate highlight the need to dissect the fundamental mechanisms that underlie normal development of the palate and how these are disturbed in cleft palate. As a result of ethical concerns, these studies cannot be performed in humans. In mice palate development mirrors that occurring in humans and the mouse is, therefore, the major animal model used in the study of palatogenesis. Although the descriptive embryology of mouse palate development is well-established, the underlying genetic events are poorly characterised.Recently, we have generated compelling evidence that the transcription factor p63, which is a master control gene, plays a central role in the genetic events driving palatal development. We have generated a complete profile of the genes expressed in the developing palate at the critical time-points in its development by using high-throughput sequencing of RNA (RNA-seq) isolated from carefully dissected mouse palates. In parallel, we have identified all the regions of the genome to which p63 binds in the developing palate using the ChIP-seq technique. Despite the generation of these genome-wide datasets and the confirmation of a large number of p63 transcriptional targets in the secondary palate, the gene regulatory networks controlled by p63 have not been elucidated. The behaviour of these complex networks cannot be predicted by simple intuitive approaches but requires sophisticated computational methods. In this project, we will firstly apply time-course methods to analyse RNA-seq data generated from cells and from palatal shelves dissected from mice in which the function of p63 has been disrupted. By combining the results from these two distinct but complementary systems, we will identify the direct and biologically-relevant early regulatory links in the p63-controlled developmental regulatory network which will facilitate our understanding of the role of p63 in secondary palate development. Secondly, we will use the data generated on the gene regulatory networks controlled by p63 to analyse a mouse which exhibits cleft palate as the result of increased p63 signalling. These experiments will allow us to determine why it is essential that the level of p63 signalling is reduced in a specific subset of cells during normal development of the palate. Finally, to facilitate collaborative working, we will ensure that all the data generated during the project are freely available via the World-Wide Web.Ultimately, the project will provide crucial information about a developmental process of major significance and serve as a test case for using systems-level developmental genetics to dissect the gene regulatory networks that are disrupted in congenital human malformations more widely.