Transcriptional regulation of liver specification in Xenopus tropicalis

描述（由申请人提供）：从多能前体细胞建立一个承诺的细胞命运需要细胞自主和非自主线索随着时间的推移协调整合。以肝脏为例，肝细胞的命运被认为是通过一系列的承诺过程获得的：细胞首先通过细胞外淋巴结信号被引导到内胚层，然后通过特异性转录因子如Gata4和FoxA1的表达获得肝脏能力，然后通过细胞外信号的作用，包括来自横隔的BMP信号和来自心脏中胚层的FGF信号，限制细胞向其他前内胚层器官发育。本研究的目的是回答肝脏命运规范中三个尚未解决的问题：哪一个节点信号的直接目标在建立肝脏能力中起作用；FoxA1的直接目标是什么？BMP和FGF调节肝脏诱导的体内机制是什么？这些问题解决了我们对肝脏发育中已知因素的转录层次的理解差距，以及肝脏发育的每个阶段如何与下一个阶段机械地联系在一起。直接回答这些问题需要大量处于早期发育阶段的胚胎，这是羊膜模型系统的局限性，此前许多关于肝脏发育的研究都是在羊膜模型系统中进行的。因此，该研究将在热带爪蟾中进行，因为热带爪蟾有大量易于操作的胚胎，适合快速筛选和生化研究。拟议的项目将产生一个代表节点信号目标的原位杂交表达模式的数据库，该数据库将提供给社区。拟议的项目还将包括培训和发展最近开发的生化和高通量测序方法的专业知识，特别是包括对现有的染色质免疫沉淀和高通量测序（ChIP-Seq）方案的改编，用于具有FoxA1抗体的热带热带热带热带热带蝗胚胎。最后，该项目将促进在热带棘球蚴进行的基础研究之间的合作，并将研究结果应用于人类胚胎干细胞定向分化到肝脏命运。这些问题的答案将详细阐述我们对肝脏器官发生的理解，并将应用于从胚胎干细胞中提取肝细胞的新方案的开发，这是最终治疗肝脏疾病的主要目标。

DESCRIPTION (provided by applicant): The establishment of a committed cell fate from a pluripotent precursor cell requires the coordinated integration of cell autonomous and non-autonomous cues through time. In the case of the liver, hepatic cell fate is thought to be acquired through a sequential process of commitment: cells are first directed to an endoderm fate by extracellular Nodal signaling, then acquire liver competence through the expression of specific transcription factors such as Gata4 and FoxA1, then are restricted from developing into other anterior endoderm organs by the action of extracellular signals including BMP signals from the septum transversum and FGF signals from the cardiac mesoderm. The goal of this research is to answer three unresolved questions in liver fate specification: which of the direct targets of Nodal signaling act in establishing liver competence; what are the direct targets of FoxA1; and what is the in vivo mechanism by which BMP and FGF regulate hepatic induction? These questions address gaps in our understanding of the transcriptional hierarchy through which known factors in liver development act, and of how each stage of liver development is mechanistically linked to the next. Answering these questions directly requires large numbers of embryos at early developmental stages, a limitation of amniote model systems where many previous studies on liver development have been conducted. Therefore, the proposed research will be carried out in the frog Xenopus tropicalis, which has large numbers of readily manipulated embryos ideal for rapid screening and biochemical investigations. The proposed project will generate a database of in situ hybridization expression patterns representing Nodal signaling targets, which will be made available to the community. The proposed project will also entail training and the development of expertise in recently-developed biochemical and high-throughput sequencing methodologies, specifically including the adaptation of existing protocols for chromatin immunoprecipitation and high throughput sequencing (ChIP-Seq) for use in X. tropicalis embryos with FoxA1 antibodies. Finally, this project will foster collaborations between basic research carried out in X. tropicalis and applications of the findings to directed differentiation of human embryonic stem cells to liver fates. The answers to these questions will elaborate our understanding of liver organogenesis, and will have applications to the development of new protocols for deriving hepatocytes from embryonic stem cells, a major goal for the eventual treatment of liver disease. PUBLIC HEALTH RELEVANCE: Understanding the steps that occur as a cell changes from a pluripotent fate to a liver fate during normal embryological development has and will continue to directly inform efforts to control the differentiation of human embryonic stem cells to liver cells. Liver cells derived from embryonic stem cells or other non-liver cell types are a potential therapeutic alternative to liver transplant for patients suffering from liver diseases. Further, a mechanistic understanding of the stages of liver development will provide insight into the genetic or developmental origins of some liver diseases arising from improper execution of the liver development program, and how they could be diagnosed or treated.